Anti-pd-l1/anti-lag3 bispecific antibodies and uses thereof

ABSTRACT

The present disclosure provides an anti-PD-L1/anti-LAG3 bispecific antibody capable to effectively block the interactions between PD-L1 and its receptor PD-1 and between LAG3 and its ligand (e.g., a MHC class II molecule and FGL1). The bispecific antibody may have high binding affinity to both of a PD-L1 protein (e.g., a human PD-L1 protein) and a LAG3 protein (e.g., a human LAG3 protein). Also provided are antibodies and fragments that have specificity to the PD-L1 or LAG3 protein alone, or antibodies and fragments having additional specificity to one or more other antigens.

BACKGROUND

Programmed death-ligand 1 (PD-L1), also known as cluster ofdifferentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a 40 kDa type 1transmembrane protein believed to play a major role in suppressing theimmune system during particular events such as pregnancy, tissueallografts, autoimmune disease and other disease states such ashepatitis. The binding of PD-L1 to PD-1 or B7.1 transmits an inhibitorysignal which reduces the proliferation of CD8+ T cells at the lymphnodes and supplementary to that PD-1 is also able to control theaccumulation of foreign antigen specific T cells in the lymph nodesthrough apoptosis which is further mediated by a lower regulation of thegene Bcl-2.

It has been shown that upregulation of PD-L1 may allow cancers to evadethe host immune system. An analysis of tumor specimens from patientswith renal cell carcinoma found that high tumor expression of PD-L1 wasassociated with increased tumor aggressiveness and an increased risk ofdeath. Many PD-L1 inhibitors are in development as immuno-oncologytherapies and are showing good results in clinical trials.

In addition to treatment of cancers, PD-L1 inhibition has also shownpromises in treating infectious diseases. In a mouse model ofintracellular infection, L. monocytogenes induced PD-L1 proteinexpression in T cells, NK cells, and macrophages. PD-L1 blockade (e.g.,using blocking antibodies) resulted in increased mortality for infectedmice. Blockade reduced TNFα and nitric oxide production by macrophages,reduced granzyme B production by NK cells, and decreased proliferationof L. monocytogenes antigen-specific CD8 T cells (but not CD4 T cells).This evidence suggests that PD-L1 acts as a positive costimulatorymolecule in intracellular infection.

Lymphocyte Activation Gene-3 (LAG-3) (also known as CD223) is a memberof the immunoglobulin (Ig) superfamily, is closely related to CD4, andvariously impacts T cell function. LAG-3 is expressed on activated Tcells, exhausted T cells, tumor infiltrating T cells, and regulatory Tcells (Tregs). Upon binding with major histocompatibility complex 2 (MHCclass II), the LAG-3/MHC class II interaction results in the negativeregulation of T cell proliferation, activation, and homeostasis.

LAG-3 represents an important immune checkpoint in cancer, similarly tocytotoxic T lymphocyte antigen-4 (CTLA-4), programmed cell deathligand-1 (PD-L1), and programmed cell death-1 (PD-1). LAG-3 not onlyexpresses on the activated/exhausted effector T cells but also onregulatory T cells. LAG3 antagonism can not only promote the activationof effector T cells, but also block the suppressive function ofregulatory T cells. Therefore, LAG-3 represents a promising target forcancer immunotherapy and preclinical evidence suggests that ananti-LAG-3 antibody can promote an anti-tumor response.

In view of the above, a need exists for developing novel agents thatmodulate the activity of LAG-3 in a manner that stimulates an immuneresponse that inhibits the growth of various cancers and tumor cells, aswell as being useful in the treatment of autoimmune, inflammatory, orviral diseases.

SUMMARY

The present disclosure provides an anti-PD-L1/anti-LAG3 bispecificantibody capable to effectively block the interactions between PD-L1 andits receptor PD-1 and between LAG3 and its ligand (e.g., a MHC class IImolecule). The bispecific antibody may have high binding affinity toboth of a PD-L1 protein (e.g., a human PD-L1 protein) and a LAG3 protein(e.g., a human LAG3 protein).

The anti-PD-L1/anti-LAG3 bispecific antibody may comprise an anti-PD-L1antibody or an antigen-binding fragment thereof as a PD-L1 targetingmoiety, which is capable of specifically recognizing and/or binding to aPD-L1 protein, and an anti-LAG3 antibody or an antigen-binding fragmentthereof as a LAG3 targeting moiety, which is capable of specificallyrecognizing and/or binding to a LAG3 protein.

The anti-PD-L1/anti-LAG3 bispecific antibody may comprise an anti-PD-L1antibody or an antigen-binding fragment thereof as a PD-L1 targetingmoiety.

In an embodiment, the anti-PD-L1 antibody or fragment thereof comprisedin the bispecific antibody can specifically bind to an immunoglobulin C(IgC) domain of PD-L1 (e.g., human PD-L1) protein. In some embodiments,the IgC domain consists of amino acid residues 133-225 of a human PD-L1protein. In some embodiments, the anti-PD-L1 antibody or fragmentthereof can bind to at least one of amino acid residues Y134, K162, andN183 of a human PD-L1 protein. In some embodiments, the anti-PD-L1antibody or fragment thereof does not bind to an immunoglobulin V (IgV)domain of the PD-L1 protein, and for example, the IgV domain consists ofamino acid residues 19-127 of a human PD-L1 protein. For example, thehuman PD-L1 protein may be selected from the group consisting ofproteins represented by GenBank Accession No. NP_001254635.1NP_001300958.1, NP_054862.1, etc., but may not be limited thereto. Theseanti-PD-L1 antibodies may be useful for therapeutic purposes such astreating various types of cancer, infections (inflammations), etc., andcan also be used for diagnostic and prognostic purposes. In anembodiment, the anti-PD-L1 antibody or fragment thereof is capable ofspecificity to a human PD-L1 protein.

The anti-PD-L1 antibody or fragment thereof may comprise (1) a VH CDR1having an amino acid sequence selected from the group consisting of SEQID NO: 1 and SEQ ID NO: 61-67; (2) a VH CDR2 having an amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:68-77, and 525-527; (3) a VH CDR3 having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 3, SEQ ID NO: 78-90 and SEQ IDNO: 513-519; (4) a VL CDR1 having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 4, SEQ ID NO: 91-92, and SEQ ID NO:520-521; (5) a VL CDR2 having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 5 and SEQ ID NO: 93-105; and (6) a VLCDR3 having an amino acid sequence selected from the group consisting ofSEQ ID NO: 6, SEQ ID NO: 106-111, and SEQ ID NO: 522-524. For example,the anti-PD-L1 antibody or fragment thereof may comprise a VH CDR1having an amino acid sequence of SEQ ID NO: 1; a VH CDR2 having an aminoacid sequence of SEQ ID NO: 2; (3) a VH CDR3 having an amino acidsequence of SEQ ID NO: 3 or 515; a VL CDR1 having an amino acid sequenceof SEQ ID NO: 4; a VL CDR2 having an amino acid sequence of SEQ ID NO:5; and a VL CDR3 having an amino acid sequence of SEQ ID NO: 6.

The anti-PD-L1/anti-LAG3 bispecific antibody may comprise an anti-LAG3antibody or an antigen-binding fragment thereof as a LAG3 targetingmoiety. In an embodiment, the anti-LAG3 antibody or fragment thereof canspecifically bind to LAG3 (e.g., human LAG3) protein; for example, theanti-LAG3 antibody or fragment thereof may bind to an extracellulardomain of LAG-3.

For instance, the anti-LAG3 antibody or fragment thereof describedherein may inhibit the binding of the LAG-3 protein to Galectin-3(LGALS3) and C-type lectin domain family 4 member G (LSECtin) protein,in addition to inhibiting the binding to MHC class II molecules, whichis a unique and considerable effect of the anti-LAG3 antibody orfragment thereof of the present disclosure, considering that existinganti-LAG-3 antibodies have only shown inhibitory effect to the bindingto MHC class II molecules. In some embodiments, the antibodies andfragments thereof of the present disclosure are capable of reversing theinhibitory effect of regulatory T cells (T_(regs)) on effector T cells(T_(effs)). In some embodiments, the antibodies and fragments thereof ofthe present disclosure are capable of inhibiting the binding betweenLAG3 Fibrinogen-like Protein 1 (FGL1).

For example, the human LAG3 protein may be selected from the groupconsisting of proteins represented by GenBank Accession No. NP_002277.4,etc., but may not be limited thereto. These anti-LAG3 antibodies may beuseful for therapeutic purposes such as treating various types ofcancer, infections (inflammations), etc., and can also be used fordiagnostic and prognostic purposes.

In an embodiment, the anti-LAG3 antibody or fragment thereof is capableof specificity to a human LAG3 protein. The anti-LAG3 antibody orfragment thereof may comprise (i) a VH CDR1 having an amino acidsequence selected from the group consisting of SEQ ID NOS: 116-117, 354,and 453-460; (ii) a VH CDR2 having an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 118-119, 355, and 461-467; (iii) aVH CDR3 having an amino acid sequence selected from the group consistingof SEQ ID NOs: 120-160, 356, and 468-475; (iv) a VL CDR1 having an aminoacid sequence selected from the group consisting of SEQ ID NOS: 163-195,229, 357, and 490; (v) a VL CDR2 having an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 196-217, 358, and 476-483; and(vi) a VL CDR3 having an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 218-228, 230-253, 359, and 484-489. Forexample, the anti-LAG3 antibody or fragment thereof may comprise a VHCDR1 having an amino acid sequence of SEQ ID NO: 354; a VH CDR2 havingan amino acid sequence of SEQ ID NO: 355 or 461; a VH CDR3 having anamino acid sequence of SEQ ID NO: 356 or 468; a VL CDR1 having an aminoacid sequence of SEQ ID NO: 357 or 490; a VL CDR2 having an amino acidsequence of SEQ ID NO: 358; and a VL CDR3 having an amino acid sequenceof SEQ ID NO: 359 or 488.

Also provided are antibodies and fragments that have specificity to thePD-L1 or LAG3 protein alone, or antibodies having additional specificityto one or more other antigens.

In one embodiment, provided is an antibody or antigen-binding fragmentthereof having specificity to a human PD-L1 protein, comprising: (1) aVH CDR1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1 and 61-67; (2) a VH CDR2 comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 2, 68-77,and 525-527; (3) a VH CDR3 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 3, 78-90, and 513-519; (4) a VLCDR1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, 91-92, and 520-521; (5) a VL CDR2 comprisingan amino acid sequence selected from the group consisting of SEQ ID NO:5, and 93-105; and (6) a VL CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 6, 106-111, and522-524.

In one embodiment, provided is an antibody or antigen-binding fragmentthereof having specificity to a human LAG3 protein, comprising: (i) a VHCDR1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 116-117, 354, and 453-460; (ii) a VH CDR2comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 118-119, 355, and 461-467; (iii) a VH CDR3 comprising anamino acid sequence selected from the group consisting of SEQ ID NO:120-160, 356, and 468-475; (iv) a VL CDR1 comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 163-195, 229,357, and 490; (v) a VL CDR2 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 196-217, 358, and 476-483; and(vi) a VL CDR3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 218-228, 230-253, 359, and 484-489.

Another embodiment provides a pharmaceutical composition comprising thebispecific antibody as described above. The pharmaceutical compositionmay further comprise a pharmaceutically acceptable carrier. Thepharmaceutical composition may be used for treating and/or preventing acancer or an infection.

Another embodiment provides a method of treating and/or preventing acancer or an infection in a subject in need thereof, comprisingadministering to the subject a pharmaceutically effective amount of thebispecific antibody or the pharmaceutical composition. The method mayfurther step of identifying the subject in need of treating and/orpreventing a cancer or an infection, prior to the administering step.

Another embodiment provides a use of the bispecific antibody or thepharmaceutical composition in treating and/or preventing a cancer or aninfection. Another embodiment provides a use of the bispecific antibodyin preparing a pharmaceutical composition for treating and/or preventinga cancer or an infection.

In the pharmaceutical compositions, methods and/or uses provided herein,the cancer may be a solid cancer or blood cancer, preferably a solidcancer.

Another embodiment provides a composition for detection of PD-L1, LAG3,or both thereof simultaneously, in a biological sample, the compositioncomprising the bispecific antibody. Another embodiment provides a methodof detection of PD-1, LAG3, or both thereof simultaneously, in abiological sample, the method comprising contacting the biologicalsample with the bispecific antibody; and detecting (measuring) anantigen-antibody reaction (binding) between the bispecific antibody andPD-1, LAG3, or both thereof.

The method of detection may further comprise, after the detecting step,determining that PD-L1, LAG3, or both thereof are present in thebiological sample when an antigen-antibody reaction is detected, and/orthat PD-L1, LAG3, or both thereof are absent (not present) in thebiological sample, when an antigen-antibody reaction is not detected.

Another embodiment provides a pharmaceutical composition for diagnosinga disease associated with PD-L1, LAG3, or both thereof, the compositioncomprising the bispecific antibody. In another embodiment, provided is ause of the bispecific antibody for diagnosing a disease associated withPD-L1, LAG3, or both thereof.

Another embodiment provides a method of diagnosing a disease associatedwith PD-L1, LAG3, or both thereof, the method comprising contacting abiological sample obtained from a patient with the bispecific antibody,and detecting antigen-antibody reaction or measuring a level ofantigen-antibody reaction in the biological sample. In some embodiments,the method may further comprise contacting a normal sample with thebispecific antibody, and measuring a level of an antigen-antibodyreaction in the normal sample. In addition, the method may furthercomprise comparing the level of the antigen-antibody reaction in thebiological sample and in the normal sample, after the measuring step. Inaddition, after the detecting step or comparing step, the method mayfurther comprise determining the patient as a patient with a diseaseassociated with PD-L1, LAG3, or both thereof, when the antigen-antibodyreaction is detected in the biological sample or the level of theantigen-antibody reaction in the biological sample is higher than thatof the normal sample.

The disease associated with PD-L1, LAG3, or both thereof may be oneassociated with activation (e.g., abnormal activation orover-activation) and/or overproduction (overexpression) of PD-1, LAG3,or both thereof. For example, the disease may be a cancer or aninfection, as described above.

An embodiment provides a polynucleotide encoding the bispecificantibody. In particular, an embodiment provides a polynucleotideencoding a heavy chain of the bispecific antibody in an IgG-scFv formwhich comprises a full-length IgG and a scFv linked to a C-terminusand/or N-terminus of the full-length IgG. Other embodiment provides apolynucleotide encoding a light chain of the bispecific antibody in anIgG-scFv form. Another embodiment provides a recombinant vectorcomprising the polynucleotide encoding a heavy chain of the bispecificantibody, the polynucleotide encoding a light chain of the bispecificantibody, or both thereof. Another embodiment provides a recombinantcell transfected with the recombinant vector.

Another embodiment provides a method of preparing the bispecificantibody, comprising expressing the polynucleotide encoding a heavychain of the bispecific antibody, the polynucleotide encoding a lightchain of the bispecific antibody in a cell. The step of expressing thepolynucleotide may be conducted by culturing the cell comprising thepolynucleotide (for example, in a recombinant vector) under a conditionallowing the expression of the polynucleotide. The method may furthercomprise isolating and/or purifying the bispecific antibody from thecell culture, after the step of expressing or culturing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that HL1210-3 can bind to human PD-L1 with high affinity.

FIG. 2 shows that HL1210-3 can efficiently inhibit the binding of humanPD-L1 to human PD1.

FIG. 3 shows the HL1210-3 antibody can highly efficiently inhibit thebinding of PD-1 on PD-L1 expressed on mammalian cells.

FIG. 4 shows that the tested anti-PD-L1 antibodies can promote human Tcell response.

FIG. 5 shows the binding kinetics of HL1210-3 to recombinant PD-1.

FIGS. 6A-6E show that all tested humanized antibodies had comparablebinding efficacy to human PD-L1 in contact to chimeric antibody.

FIGS. 7A-7C shows that all tested humanized antibodies can highefficiently bind to PD-L1 expressed on mammalian cells, comparable withchimeric antibody.

FIG. 8 shows that humanized antibody Hu1210-41 can bind to rhesus PD-L1with lower affinity and cannot bind to rat and mouse PD-L1.

FIG. 9 shows that Hu1210-41 antibody can only specifically binding toB7-H1 (PD-L1), not B7-DC, B7-1, B7-2, B7-H2, PD-1, CD28, CTLA4, ICOS andBTLA.

FIG. 10 shows that Hu1210-41 can efficiently inhibit the binding ofhuman PD-L1 to human PD1 and B7-1.

FIG. 11 shows that Hu1210-41 can efficiently inhibit the binding ofhuman PD-L1 to human PD1 and B7-1.

FIG. 12 shows that the Hu1210-8, Hu1210-9, Hu1210-16, Hu1210-17,Hu1210-21 and Hu1210-36 humanized antibodies can dose dependentlypromote the IFNγ and IL-2 production in mix lymphocyte reaction.

FIG. 13 shows that the Hu1210-40, Hu1210-41 and Hu1210-17 humanizedantibodies can dose dependently promote the IFNγ production in CMVrecall assay.

FIG. 14 shows that Hu1210-31 can inhibit the tumor growth by 30% at 5mg/kg in HCC827-NSG-xenograft model.

FIG. 15 shows that Hu1210-41 antibody can dose-dependently inhibit thetumor growth in HCC827-NSG-xenograft model, while the tumor weight wasalso dose-dependently suppressed by Hu1210-41 antibody.

FIG. 16 plots, for each PD-L1 mutant, the mean binding value as afunction of expression (control anti-PD-L1 mAb reactivity).

FIG. 17 illustrates the locations of Y134, K162, and N183, the residues(spheres) involved in binding to the anti-PD-L1 Hu1210-41 antibody.

FIG. 18 shows the results of a binding assay (to human PD-L1) for thederived antibodies.

FIG. 19 shows that antibody B6 more highly efficiently bound to PD-L1expressed on mammalian cells, as compared to the parental antibody andTecentriq™ (atezolizumab).

FIG. 20 shows the effects of the antibodies on IL2 production in Jurkatcells in which B6 also exhibited higher potency.

FIG. 21 shows that the D1-D2 domains are important for LAG-3 function.Wildtype (WT) LAG3 extracellular domain (ECD) fusion protein(LAG-3-ECD-huFc) fragments can bind to Daudi cells while D1-D2 truncatedLAG-3-ECD-huFc fragments fail to bind Daudi cells.

FIGS. 22A-22D show the binding of human anti-LAG3 antibodies to LAG3protein derived from various species. Anti-LAG-3 antibodies wereevaluated for their binding properties to human, rat, and mouse LAG3through enzyme-linked immunosorbent assay (ELISA).

FIG. 23 shows the binding of human anti-LAG3 antibodies to cell surfaceLAG-3 antigen on activated human primary CD4+ T cells. Anti-LAG-3antibodies were assessed for binding to cell surface LAG-3 antigen onactivated human primary CD4+ T cells at various concentrations (10μg/ml, 3.333 μg/ml, 1.111 μg/ml, 0.370 μg/ml, 0.123 μg/ml, 0.041 μg/ml,0.014 μg/ml and 0.005 μg/ml).

FIG. 24 shows inhibition of soluble LAG-3 (sLAG) binding to MHC class IIreceptor by anti-LAG-3 antibody. Anti-LAG-3 antibodies were evaluatedfor their ability to block the binding of sLAG-3 to MHC class IIreceptor in an in vitro binding assay using biotin-labeledLAG-3-ECD-huFcLAG-3-Fc fusion proteins and Raji cells expressing MHCclass II receptor.

FIG. 25 shows stimulation of IL-2 production in peripheral bloodmononuclear cells (PBMCs) by anti-LAG-3 antibodies. Anti-LAG-3antibodies were administrated into Staphylococcal Enterotoxin B (SEB)stimulated PBMCs at various concentrations starting from 20 μg/ml at 1:3serial dilution for 6 doses. Three days later, IL-2 concentration in theculture supernatant was evaluated by enzyme-linked immunosorbent assay(ELISA).

FIG. 26 shows Reversing the suppressive function of regulatory T cells(T_(regs)) on effector T cells (T_(effs)) using anti-LAG-3 antibodies.To evaluate the ability of anti-LAG-3 antibodies to reverse thesuppressive effect of T_(regs) on T_(effs), the antibodies of Example2.1 were used in an in vitro Tregs suppression assay.

FIGS. 27A-27C show ELISA results showing EC50 of the antibody forbinding to full extracellular domain of LAG3 (D1-D4 huFc) but not D1-D2deleted LAG3 (AD1-D2 huFc), demonstrating that 122H, 147H and 170H arepotent and selective binder for D1 and D2 domain of human LAG3.

FIGS. 28A-28C show that 122H, 147H and 170H antibodies dose dependentlyinhibited the binding of LAG3 to its receptor MHC class II molecules.

FIG. 29 shows that 122H, 147H and 170H mouse monoclonal antibodies dosedependently promoted IL2 production by Jurkat T cells.

FIG. 30 shows that Humanized monoclonal antibody 147H-13 dosedependently promoted the IL2 production by Jurkat T cells.

FIG. 31 shows binding curves of anti-LAG3 antibodies on Jurkat-LAG3cells and activated CD4 T cell.

FIG. 32 schematically illustrates an anti-PD-1/anti-LAG3 bispecificantibody according to an embodiment.

FIG. 33 shows graphs illustrating the binding of theanti-PD-L1/anti-LAG3 bispecific antibody according to an embodiment tohuman PD-L1 and human LAG3, measured by ELISA.

FIG. 34 shows the SEE assay results for the anti-PD-L1/anti-LAG3bispecific antibody according to an embodiment. It also shows graphsillustrating the T-cell promoting activities of the anti-PD-L1/anti-LAG3bispecific antibody according to an embodiment.

FIG. 35 shows a graph illustrating tumor growth inhibition effect of theanti-PD-L1/anti-LAG3 bispecific antibody according to an embodiment.

FIG. 36 shows graphs illustrating the T-cell promoting activities of theanti-PD-L1/anti-LAG3 bispecific antibody according to an embodiment.

FIG. 37 shows graphs illustrating the T-cell promoting activities of theanti-PD-L1/anti-LAG3 bispecific antibody according to an embodiment.

FIG. 38 shows the binding of anti-LAG3 monoclonal antibody B3807 andcontrol antibodies to the human LAG3 protein, through enzyme-linkedimmunosorbent assay (ELISA).

FIG. 39 shows the Biacore analysis result for B3807.

FIG. 40 shows the binding activities of B3807 to human LAG3 on Jurkatand PBMC cells.

FIG. 41 shows the inhibition of soluble LAG-3 (sLAG) binding to MHCclass II receptor by B3807.

FIG. 42 shows the effects of the B3807 on IL2 production in Jurkatcells.

FIG. 43 shows the effects of the B3807, as well as in combination withanti-PD-L1 antibody, on IL2 production in primary T cells.

FIG. 44 shows the in vivo results of B3807, alone or in combination withanti-PD-1 or anti-PD-L1 antibodies, in inhibiting tumor growth.

FIG. 45 compares B3807 and B3807b in IL2 release and cell-based bindingassays, and demonstrates their high level similarity.

FIG. 46 compares the Biacore assay results between B3807 and B3807b.

FIG. 47 demonstrates that B3807 effectively inhibited the bindingbetween soluble LAG-3 and FGL1.

DETAILED DESCRIPTION Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity for example, “an antibody,” is understood to representone or more antibodies. As such, the terms “a” (or “an”), “one or more,”and “at least one” can be used interchangeably herein.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

The term “isolated” as used herein with respect to cells, nucleic acids,such as DNA or RNA, refers to molecules separated from other DNAs orRNAs, respectively, that are present in the natural source of themacromolecule. The term “isolated” as used herein also refers to anucleic acid or peptide that is substantially free of cellular material,viral material, or culture medium when produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized. Moreover, an “isolated nucleic acid” is meant to includenucleic acid fragments which are not naturally occurring as fragmentsand would not be found in the natural state. The term “isolated” is alsoused herein to refer to cells or polypeptides which are isolated fromother cellular proteins or tissues. Isolated polypeptides is meant toencompass both purified and recombinant polypeptides.

As used herein, the term “recombinant” as it pertains to polypeptides orpolynucleotides intends a form of the polypeptide or polynucleotide thatdoes not exist naturally, a non-limiting example of which can be createdby combining polynucleotides or polypeptides that would not normallyoccur together.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences. An “unrelated” or “non-homologous” sequence sharesless than 40% identity, though preferably less than 25% identity, withone of the sequences of the present disclosure.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) has a certain percentage (for example, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” toanother sequence means that, when aligned, that percentage of bases (oramino acids) are the same in comparing the two sequences. This alignmentand the percent homology or sequence identity can be determined usingsoftware programs known in the art, for example those described inAusubel et al. eds. (2007) Current Protocols in Molecular Biology.Preferably, default parameters are used for alignment. One alignmentprogram is BLAST, using default parameters. In particular, programs areBLASTN and BLASTP, using the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalentpolynucleotides are those having the above-noted specified percenthomology and encoding a polypeptide having the same or similarbiological activity.

The term “an equivalent nucleic acid or polynucleotide” refers to anucleic acid having a nucleotide sequence having a certain degree ofhomology, or sequence identity, with the nucleotide sequence of thenucleic acid or complement thereof. A homolog of a double strandednucleic acid is intended to include nucleic acids having a nucleotidesequence which has a certain degree of homology with or with thecomplement thereof. In one aspect, homologs of nucleic acids are capableof hybridizing to the nucleic acid or complement thereof. Likewise, “anequivalent polypeptide” refers to a polypeptide having a certain degreeof homology, or sequence identity, with the amino acid sequence of areference polypeptide. In some aspects, the sequence identity is atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99%. In some aspects, theequivalent polypeptide or polynucleotide has one, two, three, four orfive addition, deletion, substitution and their combinations thereof ascompared to the reference polypeptide or polynucleotide. In someaspects, the equivalent sequence retains the activity (e.g.,epitope-binding) or structure (e.g., salt-bridge) of the referencesequence.

Hybridization reactions can be performed under conditions of different“stringency.” In general, a low stringency hybridization reaction iscarried out at about 40° C. in about 10×SSC or a solution of equivalentionic strength/temperature. A moderate stringency hybridization istypically performed at about 50° C. in about 6×SSC, and a highstringency hybridization reaction is generally performed at about 60° C.in about 1×SSC. Hybridization reactions can also be performed under“physiological conditions” which is well known to one of skill in theart. A non-limiting example of a physiological condition is thetemperature, ionic strength, pH and concentration of Mg²⁺ normally foundin a cell.

A polynucleotide is composed of a specific sequence of four nucleotidebases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil(U) for thymine when the polynucleotide is RNA. Thus, the term“polynucleotide sequence” is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching. The term “polymorphism” refers to the coexistence of morethan one form of a gene or portion thereof. A portion of a gene of whichthere are at least two different forms, i.e., two different nucleotidesequences, is referred to as a “polymorphic region of a gene.” Apolymorphic region can be a single nucleotide, the identity of whichdiffers in different alleles.

The terms “polynucleotide” and “oligonucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides or analogsthereof. Polynucleotides can have any three-dimensional structure andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, EST or SAGE tag), exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA,miRNA, recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes and primers. A polynucleotide can comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure can be impartedbefore or after assembly of the polynucleotide. The sequence ofnucleotides can be interrupted by non-nucleotide components. Apolynucleotide can be further modified after polymerization, such as byconjugation with a labeling component. The term also refers to bothdouble- and single-stranded molecules. Unless otherwise specified orrequired, any embodiment of this disclosure that is a polynucleotideencompasses both the double-stranded form and each of two complementarysingle-stranded forms known or predicted to make up the double-strandedform.

The term “encode” as it is applied to polynucleotides refers to apolynucleotide which is said to “encode” a polypeptide if, in its nativestate or when manipulated by methods well known to those skilled in theart, it can be transcribed and/or translated to produce the mRNA for thepolypeptide and/or a fragment thereof. The antisense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom.

As used herein, an “antibody” or “antigen-binding polypeptide” refers toa polypeptide or a polypeptide complex that specifically recognizes andbinds to an antigen. An antibody can be a whole antibody and any antigenbinding fragment or a single chain thereof. Thus the term “antibody”includes any protein or peptide containing molecule that comprises atleast a portion of an immunoglobulin molecule having biological activityof binding to the antigen. Examples of such include, but are not limitedto a complementarity determining region (CDR) of a heavy or light chainor a ligand binding portion thereof, a heavy chain or light chainvariable region, a heavy chain or light chain constant region, aframework (FR) region, or any portion thereof, or at least one portionof a binding protein.

The terms “antibody fragment” or “antigen-binding fragment”, as usedherein, is a portion of an antibody such as F(ab′)2, F(ab)2, Fab′, Fab,Fv, scFv and the like. Regardless of structure, an antibody fragmentbinds with the same antigen that is recognized by the intact antibody.The term “antibody fragment” includes aptamers, spiegelmers, anddiabodies. The term “antibody fragment” also includes any synthetic orgenetically engineered protein that acts like an antibody by binding toa specific antigen to form a complex.

A “single-chain variable fragment” or “scFv” refers to a fusion proteinof the variable regions of the heavy (VH) and light chains (VL) ofimmunoglobulins. In some aspects, the regions are connected with a shortlinker peptide of ten to about 25 amino acids. The linker can be rich inglycine for flexibility, as well as serine or threonine for solubility,and can either connect the N-terminus of the VH with the C-terminus ofthe VL, or vice versa. This protein retains the specificity of theoriginal immunoglobulin, despite removal of the constant regions and theintroduction of the linker. ScFv molecules are known in the art and aredescribed, e.g., in U.S. Pat. No. 5,892,019.

The term antibody encompasses various broad classes of polypeptides thatcan be distinguished biochemically. Those skilled in the art willappreciate that heavy chains are classified as gamma, mu, alpha, delta,or epsilon (γ, μ, α, δ, ε) with some subclasses among them (e.g.,γ1-γ4). It is the nature of this chain that determines the “class” ofthe antibody as IgG, IgM, IgA IgG, or IgE, respectively. Theimmunoglobulin subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgG5,etc. are well characterized and are known to confer functionalspecialization. Modified versions of each of these classes and isotypesare readily discernable to the skilled artisan in view of the instantdisclosure and, accordingly, are within the scope of the instantdisclosure. All immunoglobulin classes are clearly within the scope ofthe present disclosure, the following discussion will generally bedirected to the IgG class of immunoglobulin molecules. With regard toIgG, a standard immunoglobulin molecule comprises two identical lightchain polypeptides of molecular weight approximately 23,000 Daltons, andtwo identical heavy chain polypeptides of molecular weight53,000-70,000. The four chains are typically joined by disulfide bondsin a “Y” configuration wherein the light chains bracket the heavy chainsstarting at the mouth of the “Y” and continuing through the variableregion.

Antibodies, antigen-binding polypeptides, variants, or derivativesthereof of the disclosure include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized, primatized, or chimericantibodies, single chain antibodies, epitope-binding fragments, e.g.,Fab, Fab′ and F(ab′)2, Fd, Fvs, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv), fragments comprising either aVK or VH domain, fragments produced by a Fab expression library, andanti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto LIGHT antibodies disclosed herein). Immunoglobulin or antibodymolecules of the disclosure can be of any type (e.g., IgG, IgE, IgM,IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2)or subclass of immunoglobulin molecule.

Light chains are classified as either kappa or lambda (K, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VK) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CK) and the heavy chain (CH1, CH2 or CH3) conferimportant biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen-binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CK domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the VK domain and VH domain, or subset of the complementaritydetermining regions (CDRs), of an antibody combine to form the variableregion that defines a three dimensional antigen-binding site. Thisquaternary antibody structure forms the antigen-binding site present atthe end of each arm of the Y. More specifically, the antigen-bindingsite is defined by three CDRs on each of the VH and VK chains (i.e.CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In some instances,e.g., certain immunoglobulin molecules derived from camelid species orengineered based on camelid immunoglobulins, a complete immunoglobulinmolecule may consist of heavy chains only, with no light chains. See,e.g., Hamers-Casterman et al., Nature 363: 446-448 (1993).

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen-binding domain are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen-binding domain as the antibody assumes its threedimensional configuration in an aqueous environment. The remainder ofthe amino acids in the antigen-binding domains, referred to as“framework” regions, show less inter-molecular variability. Theframework regions largely adopt a R-sheet conformation and the CDRs formloops which connect, and in some cases form part of, the R-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen-binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable regionby one of ordinary skill in the art, since they have been preciselydefined (see www.bioinf.org.uk: Dr. Andrew C. R. Martin's Group;“Sequences of Proteins of Immunological Interest,” Kabat, E., et al.,U.S. Department of Health and Human Services, (1983); and Chothia andLesk, J. Mol. Biol., 196: 901-917 (1987)).

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaet al., J. Mol. Biol. 196: 901-917 (1987), which are incorporated hereinby reference in their entireties. The CDR definitions according to Kabatand Chothia include overlapping or subsets of amino acid residues whencompared against each other. Nevertheless, application of eitherdefinition to refer to a CDR of an antibody or variants thereof isintended to be within the scope of the term as defined and used herein.The appropriate amino acid residues which encompass the CDRs as definedby each of the above cited references are set forth in the table belowas a comparison. The exact residue numbers which encompass a particularCDR will vary depending on the sequence and size of the CDR. Thoseskilled in the art can routinely determine which residues comprise aparticular CDR given the variable region amino acid sequence of theantibody.

TABLE 1 Kabat Chothia CDR-H1 31-35 26-32 CDR-H2 50-65 52-58 CDR-H3 95-102  95-102 CDR-L1 24-34 26-32 CDR-L2 50-56 50-52 CDR-L3 89-97 91-96

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).

In addition to table above, the Kabat number system describes the CDRregions as follows: CDR-H1 begins at approximately amino acid 31 (i.e.,approximately 9 residues after the first cysteine residue), includesapproximately 5-7 amino acids, and ends at the next tryptophan residue.CDR-H2 begins at the fifteenth residue after the end of CDR-H1, includesapproximately 16-19 amino acids, and ends at the next arginine or lysineresidue. CDR-H3 begins at approximately the thirty third amino acidresidue after the end of CDR-H2; includes 3-25 amino acids; and ends atthe sequence W-G-X-G, where X is any amino acid. CDR-L1 begins atapproximately residue 24 (i.e., following a cysteine residue); includesapproximately 10-17 residues; and ends at the next tryptophan residue.CDR-L2 begins at approximately the sixteenth residue after the end ofCDR-L1 and includes approximately 7 residues. CDR-L3 begins atapproximately the thirty third residue after the end of CDR-L2 (i.e.,following a cysteine residue); includes approximately 7-11 residues andends at the sequence F or W-G-X-G, where X is any amino acid.

Antibodies disclosed herein may be from any animal origin includingbirds and mammals. Preferably, the antibodies are human, murine, donkey,rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. Inanother embodiment, the variable region may be condricthoid in origin(e.g., from sharks).

As used herein, the term “heavy chain constant region” includes aminoacid sequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain constant region comprises at least one of: aCH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region)domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof.For example, an antigen-binding polypeptide for use in the disclosuremay comprise a polypeptide chain comprising a CH1 domain; a polypeptidechain comprising a CH1 domain, at least a portion of a hinge domain, anda CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3domain; a polypeptide chain comprising a CH1 domain, at least a portionof a hinge domain, and a CH3 domain, or a polypeptide chain comprising aCH1 domain, at least a portion of a hinge domain, a CH2 domain, and aCH3 domain. In another embodiment, a polypeptide of the disclosurecomprises a polypeptide chain comprising a CH3 domain. Further, anantibody for use in the disclosure may lack at least a portion of a CH2domain (e.g., all or part of a CH2 domain). As set forth above, it willbe understood by one of ordinary skill in the art that the heavy chainconstant region may be modified such that they vary in amino acidsequence from the naturally occurring immunoglobulin molecule.

The heavy chain constant region of an antibody disclosed herein may bederived from different immunoglobulin molecules. For example, a heavychain constant region of a polypeptide may comprise a CH1 domain derivedfrom an IgG1 molecule and a hinge region derived from an IgG3 molecule.In another example, a heavy chain constant region can comprise a hingeregion derived, in part, from an IgG1 molecule and, in part, from anIgG3 molecule. In another example, a heavy chain portion can comprise achimeric hinge derived, in part, from an IgG1 molecule and, in part,from an IgG4 molecule.

As used herein, the term “light chain constant region” includes aminoacid sequences derived from antibody light chain. Preferably, the lightchain constant region comprises at least one of a constant kappa domainor constant lambda domain.

A “light chain-heavy chain pair” refers to the collection of a lightchain and heavy chain that can form a dimer through a disulfide bondbetween the CL domain of the light chain and the CH1 domain of the heavychain.

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “VH domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “CH1 domain” includes the first (most amino terminal) constantregion domain of an immunoglobulin heavy chain. The CH1 domain isadjacent to the VH domain and is amino terminal to the hinge region ofan immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat et al., U.S. Dept. of Health and Human Services, “Sequences ofProteins of Immunological Interest” (1983). The CH2 domain is unique inthat it is not closely paired with another domain. Rather, two N-linkedbranched carbohydrate chains are interposed between the two CH2 domainsof an intact native IgG molecule. It is also well documented that theCH3 domain extends from the CH2 domain to the C-terminal of the IgGmolecule and comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen-binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux et al., J. Immunol161: 4083 (1998)).

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CH1 and CK regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).

As used herein, the term “chimeric antibody” will be held to mean anyantibody wherein the immunoreactive region or site is obtained orderived from a first species and the constant region (which may beintact, partial or modified in accordance with the instant disclosure)is obtained from a second species. In certain embodiments the targetbinding region or site will be from a non-human source (e.g. mouse orprimate) and the constant region is human.

As used herein, “percent humanization” is calculated by determining thenumber of framework amino acid differences (i.e., non-CDR difference)between the humanized domain and the germline domain, subtracting thatnumber from the total number of amino acids, and then dividing that bythe total number of amino acids and multiplying by 100.

By “specifically binds” or “has specificity to,” it is generally meantthat an antibody binds to an epitope via its antigen-binding domain, andthat the binding entails some complementarity between theantigen-binding domain and the epitope. According to this definition, anantibody is said to “specifically bind” to an epitope when it binds tothat epitope, via its antigen-binding domain more readily than it wouldbind to a random, unrelated epitope. The term “specificity” is usedherein to qualify the relative affinity by which a certain antibodybinds to a certain epitope. For example, antibody “A” may be deemed tohave a higher specificity for a given epitope than antibody “B,” orantibody “A” may be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D.” Preferably, theantibody binds to an antigen (or epitope) with “high affinity”, namelywith a K_(D) of 1×10⁻⁷ M or less, more preferably 5×10⁻⁸ M or less, morepreferably 3×10⁻⁸ M or less, more preferably 1×10⁻⁸ M or less, morepreferably 25×10⁻⁹ M or less or even more preferably 1×10⁻⁹ M or less.

As used herein, the terms “treat” or “treatment” may refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the progression of cancer.Beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” mayrefer to any subject, particularly a mammalian subject, for whomdiagnosis, prognosis, or therapy is desired. Mammalian subjects includehumans, domestic animals, farm animals, and zoo, sport, or pet animalssuch as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle,cows, and so on.

As used herein, phrases such as “to a patient in need of treatment” or“a subject in need of treatment” includes subjects, such as mammaliansubjects, that would benefit from administration of an antibody orcomposition of the present disclosure used, e.g., for detection, for adiagnostic procedure and/or for treatment.

The present disclosure provides an anti-PD-L1/anti-LAG3 bispecificantibody capable to effectively block the interactions between PD-L1 andits receptor PD-1 and between LAG3 and its ligand (e.g., a MHC class 11molecule). The bispecific antibody may have high binding affinity toboth of a PD-L1 protein (e.g., a human PD-L1 protein) and a LAG3 protein(e.g., a human LAG3 protein).

The anti-PD-L1/anti-LAG3 bispecific antibody may comprise an anti-PD-L1antibody or an antigen-binding fragment thereof as a PD-L1 targetingmoiety, which is capable of specifically recognizing and/or binding to aPD-L1 protein, and an anti-LAG3 antibody or an antigen-binding fragmentthereof as a LAG3 targeting moiety, which is capable of specificallyrecognizing and/or binding to a LAG3 protein.

Anti-PD-L1 Antibody

The anti-PD-L1/anti-LAG3 bispecific antibody may comprise an anti-PD-L1antibody or an antigen-binding fragment thereof as a PD-L1 targetingmoiety. The anti-PD-L1 antibody or antigen-binding fragment thereof mayexhibit potent binding and inhibitory activities to PD-L1, and be usefulfor therapeutic and diagnostics uses.

The PD-L1 protein is a 40 kDa type 1 transmembrane protein. The PD-L1protein may be a human PD-L1 protein, and the human PD-L1 protein may beselected from the group consisting of proteins represented by GenBankAccession No. NP_001254635.1, NP_001300958.1, NP_054862.1, etc., but maynot be limited thereto. The human PD-L1 protein includes anextracellular portion including an N-terminal immunoglobulin V (IgV)domain (amino acids 19-127) and a C-terminal immunoglobulin C (IgC)domain (amino acids 133-225). Unlike pre-existing anti-PD-L1 antibodies,which bind to the IgV domain of PD-L1, thereby disrupting the bindingbetween PD-1 and PD-L1, the anti-PD-L1 antibody or fragment thereofcomprised in the bispecific antibody may not bind to an immunoglobulin V(IgV) domain of the PD-L1 protein but bind to the IgC domain of PD-L1,to effectively inhibit PD-L1, thereby improving therapeutic effects.

In particular, the anti-PD-L1 antibody or fragment thereof comprised inthe bispecific antibody can specifically bind to an immunoglobulin C(IgC) domain of PD-L1 protein. In the case of human PD-L1 protein, theIg C domain comprises or consists essentially of amino acid residues133-225 of full-length of the human PD-L1 protein. More specifically,the anti-PD-L1 antibody or fragment thereof can bind to at least oneselected from the amino acid residues Y134, K162, and N183 of humanPD-L1 protein. In some embodiments, the anti-PD-L1 antibody or fragmentthereof can bind to at least two selected from the amino acid residuesY134, K162, and N183 of human PD-L1 protein. In some embodiments, theanti-PD-L1 antibody or fragment thereof does not bind to animmunoglobulin V (IgV) domain of the PD-L1 protein, wherein the IgVdomain consists of amino acid residues 19-127 of human PD-L1 protein.

In an embodiment, antibodies and fragments thereof are provided that arecapable of specific binding to a human PD-L1 protein. These antibodiesmay be useful for therapeutic purposes such as treating various types ofcancer, infections (inflammations), etc., and can also be used fordiagnostic and prognostic purposes.

The anti-PD-L1 antibody or fragment thereof may comprise (1) a VH CDR1having an amino acid sequence selected from the group consisting of SEQID NO: 1 and SEQ ID NO: 61-67; (2) a VH CDR2 having an amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:68-77, and 525-527; (3) a VH CDR3 having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 3, SEQ ID NO: 78-90 and SEQ IDNO: 513-519; (4) a VL CDR1 having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 4, SEQ ID NO: 91-92, and SEQ ID NO:520-521; (5) a VL CDR2 having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 5 and SEQ ID NO: 93-105; and (6) a VLCDR3 having an amino acid sequence selected from the group consisting ofSEQ ID NO: 6, SEQ ID NO: 106-111, and SEQ ID NO: 522-524.

TABLE 2 CDRs of anti-PD-L1 antibodies Name Sequence SEQ ID NO: VH CDR1SY DM S   1 TYDMS  61 CYDMS  62 SFDMS  63 SHDMS  64 SWDMS  65 SYDMT  66SYDMC  67 VH CDR2 TISD G G GY IYY S D SV KG   2 TISDGGAYIYYSDSVKG  68TISDGGPYIYYSDSVKG  69 TISDGGGFIYYSDSVKG  70 TISDGGGHIYYSDSVKG  71TISDGGGWIYYSDSVKG  72 TISDGGGYIYYSDTVKG  73 TISDGGGYIYYSDCVKG  74TISDGGGYIYYSDSLKG  75 T1SDGGGYIYYSDSIKG  76 TISDGGGYIYYSDSMKG  77TISDAGGYIYYSDSVKG 525 TISDAGGYIYYRDSVKG 526 TISDGGGYIYYRDSVKG 527VH CDR3 EFGKRY ALDY   3 QFGKRYALDY  78 DFGKRYALDY  79 NFGKRYALDY  80EYGKRYALDY  81 EHGKRYALDY  82 EWGKRYALDY  83 EFAKRYALDY  84 EFPKRYALDY 85 EFGRRYALDY  86 EFGKKYALDY  87 EFGKRFALDY  88 EFGKRHALDY  89EFGKRWALDY  90 EFGKRYALDS 513 EIFNRYALDY 514 ELPWRYALDY 515 ELHFRYALDY516 ELYFRYALDY 517 ELLHRYALDY 518 ELRGRYALDY 519 VLCDR1 KA S QDVTPAVA  4 KATQDVTPAVA  91 KACQDVTPAVA  92 KAKQDVTPAVA 520 KASQDVWPAVA 521VL CDR2 STSSRY T   5 TTSSRYT  93 CTSSRYT  94 SSSSRYT  95 SMSSRYT  96SVSSRYT  97 STTSRYT  98 STCSRYT  99 STSTRYT 100 STSCRYT 101 STSSKYT 102STSSRFT 103 STSSRHT 104 STSSRWT 105 VLCDR3 QQ HYTTPLT   6 EQHYTTPLT 106DQHYTTPLT 107 NQHYTTPLT 108 QEHYTTPLT 109 QDHYTTPLT 110 QNHYTTPLT 111MQHYTTPLT 522 QQHSTTPLT 523 QQHSDAPLT 524

In some embodiments, an antibody or fragment thereof includes no morethan one, no more than two, or no more than three of the abovesubstitutions. In some embodiments, the antibody or fragment thereofincludes a VH CDR1 of SEQ ID NO: 1 or any one of SEQ ID NO: 61-67, a VHCDR2 of SEQ ID NO: 2, 525, 526 or 527, a VH CDR3 of SEQ ID NO: 3, a VLCDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ IDNO: 6.

In some embodiments, the antibody or fragment thereof includes a VH CDR1of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2 or any one of SEQ ID NO:68-77, 525, 526 or 527, a VH CDR3 of SEQ ID NO: 3, a VL CDR1 of SEQ IDNO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID NO: 6.

In some embodiments, the antibody or fragment thereof includes a VH CDR1of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, 525, 526 or 527, a VH CDR3of SEQ ID NO: 3 or any one of SEQ ID NO: 78-90 and 513-519, a VL CDR1 ofSEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID NO: 6.

In some embodiments, the antibody or fragment thereof includes a VH CDR1of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, 525, 526 or 527, a VH CDR3of SEQ ID NO: 3, a VL CDR1 of SEQ ID NO: 4 or any one of SEQ ID NO:91-92 and 520-521, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ IDNO: 6.

In some embodiments, the antibody or fragment thereof includes a VH CDR1of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, 525, 526 or 527, a VH CDR3of SEQ ID NO: 3, a VL CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5 orany one of SEQ ID NO: 93-105, and a VL CDR3 of SEQ ID NO: 6.

In some embodiments, the antibody or fragment thereof includes a VH CDR1of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, 525, 526 or 527, a VH CDR3of SEQ ID NO: 3, a VL CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5,and a VL CDR3 of SEQ ID NO: 6 or any one of SEQ ID NO: 106-111 and522-524.

For example, the anti-PD-L1 antibody or fragment thereof may comprise aVH CDR1 having an amino acid sequence of SEQ ID NO: 1; a VH CDR2 havingan amino acid sequence of SEQ ID NO: 2, 525, 526 or 527; (3) a VH CDR3having an amino acid sequence of SEQ ID NO: 3 or 515; a VL CDR1 havingan amino acid sequence of SEQ ID NO: 4; a VL CDR2 having an amino acidsequence of SEQ ID NO: 5; and a VL CDR3 having an amino acid sequence ofSEQ ID NO: 6.

In some embodiments, an anti-PD-L1 antibody or fragment thereof isprovided that comprises a VH CDR1 having an amino acid sequence of SEQID NO: 1; a VH CDR2 having an amino acid sequence of SEQ ID NO: 525; aVH CDR3 having an amino acid sequence of SEQ ID NO: 3; a VL CDR1 havingan amino acid sequence of SEQ ID NO: 4; a VL CDR2 having an amino acidsequence of SEQ ID NO: 5; and a VL CDR3 having an amino acid sequence ofSEQ ID NO: 6.

In some embodiments, an anti-PD-L1 antibody or fragment thereof isprovided that comprises a VH CDR1 having an amino acid sequence of SEQID NO: 1; a VH CDR2 having an amino acid sequence of SEQ ID NO: 526; aVH CDR3 having an amino acid sequence of SEQ ID NO: 515; a VL CDR1having an amino acid sequence of SEQ ID NO: 4; a VL CDR2 having an aminoacid sequence of SEQ ID NO: 5; and a VL CDR3 having an amino acidsequence of SEQ ID NO: 6.

Non-limiting examples of VH (heavy chain variable region) are providedin SEQ ID NOS: 7-26, 113, 493, 495, 497, 499, 501, 503, 505, 507, 509,and 511, wherein SEQ ID NO: 113 is the mouse VH, SEQ ID NOs: 7-26 arehumanized ones, and SEQ ID NO: 493, 495, 497, 499, 501, 503, 505, 507,509, and 511 is an affinity-matured one of the humanized antibodies.Further, among the humanized VHs, SEQ ID NO: 9-15, 17-21 and 23-26include one or more back-mutations to the mouse version. Likewise,non-limiting examples of VL (VK; light chain (kappa type) variableregion) are provided in SEQ ID NOS: 27-33, 494, 496, 498, 500, 502, 504,506, 508, 510, and 512. SEQ ID NO: 28 and 30 are the originally derived,CDR-grafted, and humanized sequences as shown in the examples, and SEQID NO: 29 and 31-33 are humanized VL with back-mutations.

The back-mutations may be useful for retaining certain characteristicsof the anti-PD-L1 antibodies. In some embodiments, the anti-PD-L1antibodies of the present disclosure, in particular the human orhumanized ones, may include one or more of the back-mutations. In someembodiments, the back-mutation (i.e., included amino acid at thespecified position) in a heavy chain variable region (VH) is one or moreselected from (a) Ser at position 44, (b) Ala at position 49, (c) Ala atposition 53, (d) Ile at position 91, (e) Glu at position 1, (f) Val atposition 37, (g) Thr at position 40 (h) Val at position 53, (i) Glu atposition 54, (j) Asn at position 77, (k) Arg at position 94, and (l) Thrat position 108, of the heavy chain variable region, according to Kabatnumbering, and combinations thereof. In some embodiments, the VHback-mutations are selected from (a) Ser at position 44, (b) Ala atposition 49, (c) Ala at position 53, and/or (d) Ile at position 91, ofthe heavy chain variable region, according to Kabat numbering, andcombinations thereof.

In some embodiments, the back-mutation in a light chain variable region(VL) is one or more selected from (a) Ser at position 22, (b) Gln atposition 42, (c) Ser at position 43, (d) Asp at position 60, and (e) Thrat position 63, of the light chain variable region, according to Kabatnumbering, and combinations thereof.

In some embodiments, the anti-PD-L1 antibody of the present disclosureor fragment thereof may comprise a VH selected from SEQ ID NO: 7-26,113, 493, 495, 497, 499, 501, 503, 505, 507, 509, and 511, a VL selectedfrom SEQ ID NO: 27-33, 494, 496, 498, 500, 502, 504, 506, 508, 510, and512, or their respective biological equivalents as described above. Abiological equivalent of the VH and/or VL may have an amino acidsequence that includes the designated amino acids (e.g., CDRs) whilehaving sequence identity of at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%. Abiological equivalent of SEQ ID NO: 20, for instance, can be a VH thathas an overall 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequenceidentity to SEQ ID NO: 20 but retains the CDRs (SEQ ID NO: 1-6 or theirvariants), and optionally retains one or more, or all of theback-mutations.

Non-limiting examples of the antibody or fragment thereof may comprise aheavy chain variable region comprising or consisting essentially of theamino acid sequence of SEQ ID NO: 20 or 501, or a biological equivalentthereof, and alight chain variable region comprising or consistingessentially of the amino acid sequence of SEQ ID NO: 28 or 502, or abiological equivalent thereof.

In some embodiments, the anti-PD-L1 antibody or fragment thereof furthercomprises a heavy chain constant region, a light chain constant region,an Fc region, or the combination thereof. In some embodiments, the lightchain constant region may be a kappa or lambda chain constant region. Insome embodiments, the antibody is of an isotype of IgG, IgM, IgA, IgE orIgD, for example, human IgG, human IgM, human IgA, human IgE, or humanIgD. In some embodiments, the isotype may be IgG, for example human IgG,such as, IgG1, IgG2, IgG3, or IgG4. In some embodiments, the fragment(antigen-binding fragment of the anti-PD-L1 antibody) may be anyfragment comprising heavy chain CDRs and/or light chain CDRs of theantibody, and for example, it may be selected from the group consistingof Fab, Fab′, F(ab′)₂, Fd (comprising a heavy chain variable region anda CH1 domain), Fv (a heavy chain variable region and/or a light chainvariable region), single-chain Fv (scFv; comprising or consistingessentially of a heavy chain variable region and a light chain variableregion, in any order, and a peptide linker between the heavy chainvariable region and the light chain variable region), single-chainantibodies, disulfide-linked Fvs (sdFv), and the like.

Without limitation, the anti-PD-L1 antibody or fragment thereof is achimeric antibody, a humanized antibody, or a fully human antibody. Inone aspect, antibody or fragment thereof is not naturally occurring, orchemically or recombinantly synthesized.

Given that each of these antibodies can bind to PD-L1 such as humanPD-L1, the CDR sequences or VH and VL sequences can be “mixed andmatched” to create other anti-LAG-3 binding molecules of the disclosure.Preferably, when the CDR sequences or VH and VL chains are mixed andmatched, for example, a VH sequence from a particular V_(H)/V_(L)pairing is replaced with a structurally similar VH sequence. Likewise,preferably a VL sequence from a particular V_(H)/V_(L) pairing isreplaced with a structurally similar VL sequence.

Anti-LAG3 Antibody

The anti-PD-L1/anti-LAG3 bispecific antibody may comprise an anti-LAG3antibody or an antigen-binding fragment thereof as a LAG3 targetingmoiety.

In an embodiment, antibodies and fragments thereof are provided that canspecifically bind to LAG3 (e.g., human LAG3) protein; for example, theanti-LAG3 antibody or fragment thereof may bind to an extracellulardomain of LAG-3.

For example, the human LAG3 protein may be selected from the groupconsisting of proteins represented by GenBank Accession No. NP_002277.4,etc., but is may not be limited thereto. These anti-LAG3 antibodies maybe useful for therapeutic purposes such as treating various types ofcancer, infections (inflammations), etc., and can also be used fordiagnostic and prognostic purposes.

The term “LAG-3” or “LAG3” refers to Lymphocyte Activation Gene-3. TheLAG3 protein, which belongs to immunoglobulin (Ig) superfamily,comprises a 503-amino acid type I transmembrane protein with fourextracellular Ig-like domains, designated D1 to D4. As described herein,the term “LAG-3” includes variants, isoforms, homologs, orthologs, andparalogs. For example, antibodies specific for a human LAG-3 proteinmay, in certain cases, cross-react with a LAG-3 protein from a speciesother than human. In other embodiments, the antibodies specific for ahuman LAG-3 protein may be completely specific for the human LAG-3protein and may not exhibit species or other types of cross-reactivity,or may cross-react with LAG-3 from certain other species but not allother species (e.g., cross-react with monkey LAG-3, but not mouseLAG-3). The term “human LAG-3” refers to human sequence LAG-3, such asthe complete amino acid sequence of human LAG-3 having GenBank AccessionNo. NP 002277.4. The term “mouse LAG-3” refers to mouse sequence LAG-3,such as the complete amino acid sequence of mouse LAG-3 having GenBankAccession No. NP 032505. LAG-3 is also known in the art as, for example,CD223. The human LAG-3 sequence may differ from human LAG-3 of GenBankAccession No. NP 002277.4 by having, e.g., conserved mutations ormutations in non-conserved regions and the LAG-3 has substantially thesame biological function as the human LAG-3 of GenBank Accession No. NP002277.4. For example, a biological function of human LAG-3 is having anepitope in the extracellular domain of LAG-3 that is specifically boundby an antibody of the instant disclosure or a biological function ofhuman LAG-3 is binding to MHC Class II molecules.

As demonstrated in the experimental examples, some of the anti-LAG-3antibodies disclosed herein exhibited activities not shown with knownanti-LAG-3 antibodies. For instance, the presently disclosed antibodiesmay inhibit the binding of the LAG-3 protein to Galectin-3 (LGALS3) andC-type lectin domain family 4 member G (LSECtin) protein, in addition tothe binding to MHC class II molecules. Known anti-LAG-3 antibodies, bycontrast, have only shown inhibitory effect to the binding to MHC classII molecules. In some embodiments, the antibodies and fragments thereofof the present disclosure are capable of reversing the inhibitory effectof regulatory T cells (T_(regs)) on effector T cells (T_(effs)). In someembodiments, the antibodies and fragments thereof of the presentdisclosure are capable of inhibiting the binding between LAG3 andFibrinogen-like Protein 1 (FGL1).

These anti-LAG3 antibodies may be useful for therapeutic purposes suchas treating various types of cancer, infections (inflammations), etc.,and can also be used for diagnostic and prognostic purposes.

In an embodiment, an antibody or fragment thereof is provided that iscapable of specificity to a human LAG3 protein. The anti-LAG3 antibodyor fragment thereof may comprise (i) a VH CDR1 having an amino acidsequence selected from the group consisting of SEQ ID NOS: 116-117, 354,and 453-460; (ii) a VH CDR2 having an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 118-119, 355, and 461-467; (iii) aVH CDR3 having an amino acid sequence selected from the group consistingof SEQ ID NOs: 120-160, 356, and 468-475; (iv) a VL CDR1 having an aminoacid sequence selected from the group consisting of SEQ ID NOS: 163-195,229, 357, and 490; (v) a VL CDR2 having an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 196-217, 358, and 476-483: and(vi) a VL CDR3 having an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 218-228, 230-253, 359, and 484-489. Forexample, the anti-LAG3 antibody or fragment thereof may comprise a VHCDR1 having an amino acid sequence of SEQ ID NO: 354; a VH CDR2 havingan amino acid sequence of SEQ ID NO: 355 or 461; a VH CDR3 having anamino acid sequence of SEQ ID NO: 356 or 468; a VL CDR1 having an aminoacid sequence of SEQ ID NO: 357 or 490; a VL CDR2 having an amino acidsequence of SEQ ID NO: 358; and a VL CDR3 having an amino acid sequenceof SEQ ID NO: 359 or 488.

TABLE 3 CDRs of anti-LAG3 antibodies Name Sequence SEQ ID NO: VH CDR1SYAIS 116 SYAMS 117 GYTFTNYWLG 354 GYTFENYWLG 453 GYMFTNYWLG 454GYTFDNYWLG 455 GYTFGNYWLG 456 GYTFTNYWLW 457 GYLFTNYWLG 458 GYTFTNYWLS459 GFTFTNYWLG 460 VH CDR2 GIIPIFGTANYAQKFQG 118 AISGSGGSTYYADSVKG 119DIYPGGDYINYNEKFKG 355 DIYPGGDYIVYNEKFKG 461 DIYPGGDIINYNEKFKG 462DIYPGGDVINYNEKFKG 463 DIFPGGDYINYNEKFKG 464 DIYPGGDLINYNEKFKG 465DIYPGGDHINYNEKFKG 466 EIYPGGDYITYNEKFKG 467 VH CDR3 ARGSSWFDY 120ASSYHGGGYHRY 121 TTSKYSGSALRY 122 ARDRTGAIDY 123 ARHETVAGSFDY 124ARTGYYGGNSGAFDI 125 ARAGTGMDLVFNS 126 ARGLARGDLNFGY 127 TREPHFDY 128TTAAPGSYYLVFHY 129 ARDAGPVGYYGMDV 130 AGDGLYGSGSFGY 131 AKDIRWFYGMDV 132ARHESGIAGGHFDY 133 AKDIRWYYGMDV 134 AKGVRGTYQIGYYGMDV 135 ARQGTAMALDY136 VRDLQDWNYGGAAY 137 ARDDYYYGQFDS 138 AREITGTSYTALDS 139 ARGHIDGQAAGDY140 AASTLRVPNPPY 141 ARSGDRYDIAVSGY 142 TRGQDSTWYSSFDY 143 AASTLRLPNPPY144 ATTQTSFTSHGMDV 145 ARVRKTPFWGALDS 146 ARGFTYGDFIFDY 147ARDVRGVTYLGMDV 148 ARVRKTPFWGTLDS 149 ARVRRTPFWGALDS 150AKRKGLGSPTDYYYGMDV 151 VRPEYDTYYYGMDV 152 AKGGGSYDY 153 ARALNGMDV 154TRPLQGIAAADSYYYYAMDV 155 ARLHSYLSEEFDP 156 AKLSAVNTYIDD 157ARVTKTPFWGTLDY 158 ARVSQSPVWGYIDY 159 AKDGYYDFWSGYSDY 160 PNLPGDY 356PNLPKDH 468 PDLPGDY 469 PGLPKDY 470 PNLPKDY 471 PNLPRDY 472 PGLPRDY 473PGLPQDY 474 PDLPKDY 475 VL CDR1 QANQDIHHYLN 161 KSSQSVLYSSSNKNYLA 162KSSQSVLYSSNNKNYLA 163 RSSQNLLHSDGYNYLN 164 KSSQSVLYTSNNKNYLA 165QASQDINRYLS 166 QASQDISNYLN 167 QASQDISNYLN 167 RASQTISSHLN 168RASQGIAGWLA 169 RASQGVSSWLA 170 KSSQSLFYHSNNHNYLA 171 RASQGISSSLA 172QASRDISNSLS 173 RASQSISRYLN 174 RASRSISNWLA 175 KSSQSVFYRSNQKNYLA 176RASQSVSSYLA 177 RASRGISSWLA 178 RASQGISSWLA 179 RASQSISSYLN 180RASQAISNLLA 181 RASQGISTWLA 182 RASQGIASNLA 183 RASQGVSSYLA 184RASQSIYTYLN 185 RASQFVSDWLA 186 RASQTISTWLA 187 RASQGISSYLA 188RASQSIGYWLA 189 RATQSISSWLA 190 RASQGVRNWLA 191 RASQSINNYLA 192RASQDITSWLA 193 RASQGIYDYLA 194 RASEGISGWLA 195 RASQDIVNWLA 229RSSKSLLHSNGITYLY 357 RSSKSLLHS Q GITYLY 490 VL CDR2 DASILQS 196 WASTRES197 LGSNRAT 198 DASNLET 199 AASSLQS 200 AASTLQS 201 AAFSLQS 202 GASSRAT203 GISSRAT 204 AVSTLQS 205 DISTLQN 206 GASTLQS 207 GASSLQS 208 AASTLES209 DASSLQS 210 KASNLQS 211 TASTLQN 212 RASSLQS 213 AASHLQS 214 DASTLQS215 AASNLER 216 AASSLET 217 QVSNLAS 358 QVSNLAR 476 QKSNLAS 477 QVSNLAV478 QVSNLAL 479 QVDNLAS 480 QVSNLAT 481 HVSNLAS 482 QVSNRAS 483 VL CDR3QQADSFPIT 218 QQSYSTPWT 219 QQYYSTPWT 220 QQSFTTPWT 221 QQYDNLPPT 222QQSYGSPVT 223 QQGNSFPFT 224 QQAKSFPLT 225 QQVKSFPLT 226 QQYYNTPWT 227QQTKNFPLT 228 QQTKSFPLT 230 QQSYNTPRT 231 QQSYRAPWT 232 QQANNFPLT 233QQGNSFPLT 234 QQSKNFPVT 235 QQANSFPLT 236 QQLESYPLT 237 QQYYSSPT 238QQLKTFPLT 239 QQTNWFPLT 240 QQAQSFPIT 241 QQAHSFPLT 242 LQDYHFPLT 243QQGHSFPLT 244 QQSYIFPLT 245 QQYDTYWT 246 QQLNSYPLFT 247 QQYSSYWT 248LQHNTYPFT 249 QQGHSFPLT 250 QQAHSFPFT 251 QQANMFPLT 252 QQADSFPFT 253AQNLELPWT 359 GQNLELPWT 484 AQNLEMPWT 485 GQNLEMPWT 486 AQYLEEPWT 487AQYLELPWT 488 GQYLELPWT 489

In non-limiting examples, the antibody or fragment having specificity toLAG3 has a combination of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2,and VL CDR3 as shown in any of the antibodies listed in Table 27. Forinstance, the CDRs can be those from 147H 3807, which include a VH CDR1of SEQ ID NO:354, a VH CDR2 of SEQ ID NO:461, a VH CDR3 of SEQ IDNO:468, a VL CDR1 of SEQ ID NO:490, a VL CDR2 of SEQ ID NO:358, and a VLCDR3 of SEQ ID NO:488. Variants of these antibodies are also provided,such as those having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 99.5%sequence identity to the heavy chain/light chain variable regions andretaining the respective CDR sequences.

In one embodiment, for instance, provided is an antibody orantigen-binding fragment thereof, having specificity to a human LAG3protein and comprising: a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:443, or a polypeptide having at least90% sequence identity to the amino acid sequence of SEQ ID NO:443 andhaving a VH CDR1 comprising the amino acid sequence of SEQ ID NO:354, aVH CDR2 comprising the amino acid sequence of SEQ ID NO:461, and a VHCDR3 comprising the amino acid sequence of SEQ ID NO:468, and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:444, or a polypeptide having at least 90% sequence identity to theamino acid sequence of SEQ ID NO:444 and having a VL CDR1 comprising theamino acid sequence of SEQ ID NO:490, a VL CDR2 comprising the aminoacid sequence of SEQ ID NO:358, and a VL CDR3 comprising the amino acidsequence of SEQ ID NO:488.

In non-limiting examples of the anti-LAG3 antibody or fragment thereof,

(1) the heavy chain variable region may comprise or consist essentiallyof a polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 254-302, 352, 360-373, 375, 377, 379, 381,383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409,411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437,439, 441, 443, 445, 447, 449, 451 and 491, or a polypeptide having asequence identity of at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% to theabove described amino acid sequences; and/or

(2) the light chain variable region may comprise or consist essentiallyof a polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 303-351, 353, 374, 376, 378, 380, 382, 384,386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412,414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440,442, 444, 446, 448, 450, 452 and 492, or a polypeptide having a sequenceidentity of at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% to the abovedescribed amino acid sequences.

Non-limiting examples of the anti-LAG3 antibody or fragment thereof maycomprise a heavy chain variable region comprising or consistingessentially of the amino acid sequence of SEQ ID NO: 352 or 443 and alight chain variable region comprising or consisting essentially of theamino acid sequence of SEQ ID NO: 353 or 444.

For a humanized antibody or fragment, certain back mutations can beincorporated. In some embodiments, the heavy chain variable regioncomprises one or more amino acid residues selected from the groupconsisting of:

(a) Ala (A) at position 71,

(b) Leu (L) at position 69,

(c) Lys (K) at position 66,

(d) Ala (A) at position 67,

(e) Ile (I) at position 48,

(f) Ile (I) at position 37,

(g) Lys (K) at position 38,

(h) Phe (F) at position 91, and

(i) Glu (E) at position 1, according to Kabat numbering, andcombinations thereof.

In some embodiments, the heavy chain variable region comprises Ala (A)at position 71. In some embodiments, the heavy chain variable regioncomprises Leu (L) at position 69. In some embodiments, the heavy chainvariable region comprises Lys (K) at position 66. In some embodiments,the heavy chain variable region comprises Ala (A) at position 67. Insome embodiments, the heavy chain variable region comprises Ile (I) atposition 48. In some embodiments, the heavy chain variable regioncomprises Ile (I) at position 37. In some embodiments, the heavy chainvariable region comprises Lys (K) at position 38. In some embodiments,the heavy chain variable region comprises Phe (F) at position 91. Insome embodiments, the heavy chain variable region comprises Glu (E) atposition 1.

In some embodiments, the heavy chain variable region comprises one ormore amino acid residues selected from the group consisting of

(a) Ala (A) at position 71,

(b) Leu (L) at position 69,

(c) Lys (K) at position 66,

(d) Ala (A) at position 67,

(e) Ile (I) at position 48,

(f) Ile (I) at position 37, and

(g) Lys (K) at position 38, according to Kabat numbering, andcombinations thereof. In some embodiments, the heavy chain variableregion comprises all of the above recited residues.

The antibodies of the disclosure are characterized by particularfunctional features or properties of the antibodies. For example, theantibodies specifically bind to human LAG-3 and may bind to LAG-3 fromcertain other species, e.g., monkey LAG-3, e.g., cynomolgus monkey,rhesus monkey, but may not substantially bind to LAG-3 from certainother species, e.g., mouse LAG-3. Preferably, an antibody of thedisclosure binds to human LAG-3 with high affinity.

The ability of the antibody to stimulate an immune response, such as anantigen-specific T cell response, can be indicated by, for example, theability of the antibody to stimulate interleukin-2 (IL-2) or interferongamma (IFN-gamma) production in an antigen-specific T cell response. Incertain embodiments, an antibody of the disclosure binds to human LAG-3and exhibits an ability to stimulate an antigen-specific T cellresponse. In other embodiments, an antibody of the disclosure binds tohuman LAG-3 but does not exhibit an ability to stimulate anantigen-specific T cell response. Other means by which to evaluate theability of the antibody to stimulate an immune response include theability of the antibody to inhibit tumor growth, such as in an in vivotumor graft model or the ability of the antibody to stimulate anautoimmune response, such as the ability to promote the development ofan autoimmune disease in an autoimmune model, such as the ability topromote the development of diabetes in the NOD mouse model.

The binding of an antibody of the disclosure to LAG-3 can be assessedusing one or more techniques well established in the art. For example,in a preferred embodiment, an antibody can be tested by a flow cytometryassay in which the antibody is reacted with a cell line that expresseshuman LAG-3, such as CHO cells that have been transfected to expressLAG-3, e.g., human LAG-3, or monkey LAG-3, e.g., rhesus or cynomolgusmonkey or mouse LAG-3 on their cell surface. Other suitable cells foruse in flow cytometry assays include anti-CD3-stimulated CD4⁺ activatedT cells, which express native LAG-3. Additionally, or alternatively, theis binding of the antibody, including the binding kinetics (e.g., K_(D)value) can be tested in BIAcore binding assays. Still other suitablebinding assays include ELISA assays, for example using a recombinantLAG-3 protein. Preferably, an antibody of the disclosure binds to aLAG-3 protein with a K_(D) of 5×10⁻⁸ M or less, binds to a LAG-3 proteinwith a K_(D) of 2×10⁻⁸ M or less, binds to a LAG-3 protein with a K_(D)of 5×10⁻⁹ M or less, binds to a LAG-3 protein with a K_(D) of 4×10⁻⁹ Mor less, binds to a LAG-3 protein with a K_(D) of 3×10⁻⁹ M or less,binds to a LAG-3 protein with a K_(D) of 2×10⁻⁹ M or less, binds to aLAG-3 protein with a K_(D) of 125×10⁻⁹ M or less, binds to a LAG-3protein with a K_(D) of 5×10⁻¹⁰ M or less, or binds to a LAG-3 proteinwith a K_(D) of 1×10⁻¹⁰ M or less.

In some embodiments, the anti-LAG3 antibody or fragment thereof furthercomprises a heavy chain constant region, a light chain constant region,an Fc region, or the combination thereof. In some embodiments, the lightchain constant region may be a kappa or lambda chain constant region. Insome embodiments, the antibody is of an isotype of IgG, IgM, IgA, IgE orIgD, for example, human IgG, human IgM, human IgA, human IgE, or humanIgD. In some embodiments, the isotype may be IgG, for example human IgG,such as, IgG1, IgG2, IgG3, or IgG4. In some embodiments, the fragment(antigen-binding fragment of the anti-PD-L1 antibody) may be anyfragment comprising heavy chain CDRs and/or light chain CDRs of theantibody, and for example, it may be selected from the group consistingof Fab, Fab′, F(ab′)₂, Fd (comprising a heavy chain variable region anda CH1 domain), Fv (a heavy chain variable region and/or a light chainvariable region), single-chain Fv (scFv; comprising or consistingessentially of a heavy chain variable region and a light chain variableregion, in any order, and a peptide linker between the heavy chainvariable region and the light chain variable region), single-chainantibodies, disulfide-linked Fvs (sdFv), and the like.

Without limitation, the anti-LAG3 antibody or fragment thereof is achimeric antibody, a humanized antibody, or a fully human antibody. Inone aspect, antibody or fragment thereof is not naturally occurring, orchemically or recombinantly synthesized.

Given that each of these antibodies can bind to LAG-3 such as humanLAG-3, the CDR sequences or the VH and VL sequences can be “mixed andmatched” to create other anti-LAG-3 binding molecules of the disclosure.Preferably, when the CDRs sequences or VH and VL chains are mixed andmatched, for example, a VH sequence from a particular V_(H)/V_(L)pairing is replaced with a structurally similar VH sequence. Likewise,preferably a VL sequence from a particular V_(H)/V_(L) pairing isreplaced with a structurally similar VL sequence.

Anti-PD-L1/Anti-LAG3 Bispecific Antibody

In the bispecific antibody comprising the PD-L1 targeting moiety and theLAG3 targeting moiety, one of the PD-L1 targeting moiety and the LAG3targeting moiety can be a full-length antibody, and the other can be anantigen-binding fragment (e.g., scFv) comprising heavy chain CDRs, lightchain CDRs, or a combination thereof. The full-length antibody targetingone of PD-L1 and LAG3 proteins, and the antigen-binding fragmenttargeting the other protein may be chemically linked (e.g., covalentlylinked) directly or via a peptide linker. The antigen-binding fragment(e.g., scFv) may be linked directly or via a peptide linker toN-terminus of the full-length antibody (e.g., N-terminus of a lightchain or a heavy chain of the full-length antibody), C-terminus of thefull-length antibody (e.g., C-terminus of a heavy chain (or Fc or CH3domain) of the full-length antibody), or both thereof (see FIG. 32).

In an embodiment, the bispecific antibody may comprise a full-lengthanti-PD-L1 antibody, an antigen-binding fragment (e.g., scFv) of ananti-LAG3 antibody, and a peptide linker therebetween. In otherembodiment, the bispecific antibody may comprise a full-length anti-LAG3antibody, an antigen-binding fragment (e.g., scFv) of an anti-PD-L1antibody, and a peptide linker therebetween.

In an embodiment, the scFv contained in the bispecific antibody maycomprise a heavy chain variable region and a light chain variable regionin any order. For example, the scFv contained in the bispecific antibodymay comprise a heavy chain variable region and a light chain variable,in a direction from N-terminus to C-terminus, and optionally a peptidelinker therebetween, or alternatively, the scFv contained in thebispecific antibody may comprise a light chain variable region and aheavy chain variable, in a direction from N-terminus to C-terminus, andoptionally a peptide linker therebetween.

The use of a peptide linker for the bispecific antibody may lead to ahigh purity of the antibody.

As used herein, the term “peptide linker” may be those including anyamino acids of 1 to 100, particularly 2 to 50, and any kinds of aminoacids may be included without any restrictions. The peptide linker mayinclude for example, Gly, Asn and/or Ser residues, and also includeneutral amino acids such as Thr and/or Ala. Amino acid sequencessuitable for the peptide linker may be those known in the relevant art.Meanwhile, a length of the peptide linker may be variously determinedwithin such a limit that the functions of the fusion protein will not beaffected. For instance, the peptide linker may be formed by including atotal of about 1 to about 100, about 2 to about 50, or about 5 to about25 of one or more selected from the group consisting of Gly, Asn, Ser,Thr, and Ala. In one embodiment, the peptide linker may be representedas (G_(m)S_(l))_(n) (m, l, and n, are independently an integer of about1 to about 10, particularly an integer of about 2 to about 5). Forexample, the examples of the peptide liners are summarized as follows:

Examples Linker Linker Function Fusion Protein Type Sequence^(a) Ref.Increase scFv flexible (GGGGS)₃ [46] Stabilitiy/Folding G-CSF-Tfflexible (GGGGS)₃ [20] HBsAg preS1 flexible (GGGGS)₃ [85] Myc- Est2pflexible (Gly)₈ [30] albumin-ANF flexible (Gly)₆ [31] virus coat proteinrigid (EAAAK)₃ [50] beta-glucanase-xylanase rigid (EAAAK)_(a )(n = 1-3)[52] Increase hGH-Tf and Tf-hGH rigid A(EAAAK)_(a)ALEA(EAAAK)_(a)A [18]expression G-CSF-Tf and rigid A(EAAAK)_(a)ALEA(EAAAK)_(a)A [18] Tf-G-CSFImprove G-CSF-Tf flexible (GGGGS)₃ [20] biological G-CSF-Tf rigidA(EAAAK)_(a)ALEA(EAAAK)_(a)A [20] activity hGH-Tf rigidA(EAAAK)_(a)ALEA(EAAAK)_(a)A [40] HSA-IFN-α2b flexible GGGGS [17]HSA-IFN-α2b rigid PAPAP [17] HSA-IFN-α2b rigid AEAAAKEAAAKA [17]PGA-rTHS flexible (GGGGS)_(a )(n = 1, 2, 4) [55] interferon- -gp120rigid (Ala-Pro)_(a )(10-34 aa) [54] GSF-S-S-TY cleavable disulfide [39]IFN-α2b-HSA cleavable disulfide [42] Enable targeting FIX-albumincleavable VSQTSKLTR AETVFPDV^(b) [59] LAP-IFN- cleavable PLG LWA ^(c)[64] MazE-MazF cleavable RVL AEA; EDVVCC SMSY; [68] GGIEGR GS^(c)Immunotoxins cleavable TRHQPR GWE; [72] AGNRVRR SVG; RRRRRRR R R^(d)Immunotoxin cleavable GFLG^(c) [77] Alter PK G-CSF-Tf and hGH-Tfdipeptide LE [79] rigid A(EAAAK)_(a)ALEA(EAAAK)_(a)A cleavable Disulfide

In another embodiment, both of the PD-L1 targeting moiety and the LAG3targeting moiety may be a full-length antibody or an antigen-bindingfragment comprising heavy chain CDRs, light chain CDRs, or a combinationthereof.

In another embodiment, the bispecific antibody may be in a heterodimericform, which comprises a first arm including a pair of a first heavychain and a first light chain targeting one of PD-L1 and LAG3, and asecond arm including a pair of a second heavy chain and a second lightchain targeting the other one.

In an embodiment, the full-length antibody may be in a full-lengthimmunoglobulin form (e.g., IgG, IgM, IgA, IgE or IgD, such as, humanIgG, human IgM, human IgA, human IgE, or human IgD), and theantigen-binding fragment may be selected from the group consisting ofFab, Fab′, F(ab′)₂, Fd, Fv, scFv, single-chain antibodies, sdFv, and thelike, as described above. For example, the full-length antibody may bein a full-length human IgG (human IgG1, human IgG2, human IgG3, or humanIgG4) form, and the antigen-binding fragment may be scFv.

For example, an antibody described herein may comprise a flexible linkersequence, or may be modified to add a functional moiety (e.g., PEG, adrug, a toxin, or a label).

In some embodiments, a bi- or multi-specific antibody is provided, whichincludes anti-PD-L1 antibody or an antigen-binding fragment thereof andan anti-LAG3 antibody or an antigen-binding fragment thereof, whereinthe anti-PD-L1 antibody or antigen-binding fragment thereof is capableof specifically binding to an immunoglobulin C (Ig C) domain of a humanProgrammed death-ligand 1 (PD-1) protein, wherein the Ig C domainconsists of amino acid residues 133-225; and the anti-LAG3 antibody orantigen-binding fragment thereof is capable of binding to a MHC class IImolecule and/or FGL1.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragmentthereof includes a VH CDR1 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 1 and 61-67; a VH CDR2comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, 68-77, and 525-527; a VH CDR3 comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 3, 78-90, and513-519; a VL CDR1 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 4, 91-92, and 520-521; a VL CDR2comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 5, and 93-105; and a VL CDR3 comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 6, 106-111,and 522-524, and the anti-LAG3 antibody or antigen-binding fragmentthereof includes a VH CDR1 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 116-117, 354, and 453-460; a VHCDR2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 118-119, 355, and 461-467; a VH CDR3 comprisingan amino acid sequence selected from the group consisting of SEQ ID NO:120-160, 356, and 468-475; a VL CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 163-195, 229, 357, and490; a VL CDR2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 196-217, 358, and 476-483; and a VL CDR3comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 218-228, 230-253, 359, and 484-489.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragmentthereof includes a VH CDR1 having an amino acid sequence of SEQ ID NO:1; a VH CDR2 having an amino acid sequence of SEQ ID NO: 525; a VH CDR3having an amino acid sequence of SEQ ID NO: 3; a VL CDR1 having an aminoacid sequence of SEQ ID NO: 4; a VL CDR2 having an amino acid sequenceof SEQ ID NO: 5; and a VL CDR3 having an amino acid sequence of SEQ IDNO: 6, and the anti-LAG3 antibody or antigen-binding fragment thereofincludes a VH CDR1 comprising the amino acid sequence of SEQ ID NO:354,a VH CDR2 comprising the amino acid sequence of SEQ ID NO:461, a VH CDR3comprising the amino acid sequence of SEQ ID NO:468, a VL CDR1comprising the amino acid sequence of SEQ ID NO:490, a VL CDR2comprising the amino acid sequence of SEQ ID NO:358, and a VL CDR3comprising the amino acid sequence of SEQ ID NO:488.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragmentthereof includes a VH CDR1 having an amino acid sequence of SEQ ID NO:1; a VH CDR2 having an amino acid sequence of SEQ ID NO: 526; a VH CDR3having an amino acid sequence of SEQ ID NO: 515; a VL CDR1 having anamino acid sequence of SEQ ID NO: 4; a VL CDR2 having an amino acidsequence of SEQ ID NO: 5; and a VL CDR3 having an amino acid sequence ofSEQ ID NO: 6, and the anti-LAG3 antibody or antigen-binding fragmentthereof includes a VH CDR1 comprising the amino acid sequence of SEQ IDNO:354, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:461, aVH CDR3 comprising the amino acid sequence of SEQ ID NO:468, a VL CDR1comprising the amino acid sequence of SEQ ID NO:490, a VL CDR2comprising the amino acid sequence of SEQ ID NO:358, and a VL CDR3comprising the amino acid sequence of SEQ ID NO:488. Antibodies orvariants described herein may comprise derivatives that are modified,e.g., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody from bindingto the antigen (e.g., an epitope). For example, but not by way oflimitation, the antibodies can be modified, e.g., by at least oneselected from the group consisting of glycosylation, acetylation,pegylation, phosphorylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to acellular ligand or other protein, and the like. Any of numerous chemicalmodifications may be carried out by known techniques, including, but notlimited to specific chemical cleavage, acetylation, formylation,metabolic synthesis of tunicamycin, etc. Additionally, the antibodiesmay contain one or more non-classical amino acids.

The antibodies or fragments thereof can be detectably labeled by tagging(coupling) with a conventional labeling material selected fromchemiluminescent compounds, fluorescent compounds (e.g., fluorescenceemitting metals), radioisotopes, dyes, etc. The presence of the taggedantibodies or fragments thereof can be detected by measuring a signalarising during a chemical reaction between the is antibody (or fragmentthereof) and the labeling material. Examples of particularly usefullabeling material may be at least one selected from the group consistingof luminol, isoluminol, theromatic acridinium ester, imidazole,acridinium salt, oxalate ester, fluorescence emitting metals, and thelike. For example, the fluorescence emitting metals may be ¹⁵²Eu, orothers of the lanthanide series. These metals can be attached to theantibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

In certain embodiments, the prepared bispecific antibodies will notelicit a deleterious immune response in the animal to be treated, e.g.,in a human. In one embodiment, the bispecific antibody may be modifiedto reduce their immunogenicity using any conventional techniques. Forexample, the bispecific antibody may be a humanized, primatized,deimmunized, or chimeric antibody. These types of antibodies are derivedfrom a non-human antibody, typically a murine or primate antibody, thatretains or substantially retains the antigen-binding properties of theparent antibody, but which is less immunogenic in humans. This may beachieved by various methods, including (a) grafting the entire non-humanvariable domains onto human constant regions to generate chimericantibodies; (b) grafting at least a part of one or more of the non-humancomplementarity determining regions (CDRs) into a human framework andconstant regions with or without retention of critical frameworkresidues; or (c) transplanting the entire non-human variable domains,but “cloaking” them with a human-like section by replacement of surfaceresidues.

De-immunization can also be used to decrease the immunogenicity of anantibody. As used herein, the term “de-immunization” may includealteration of an antibody to modify T-cell epitopes (see, e.g.,International Application Publication Nos.: WO/9852976 A1 and WO/0034317A2). For example, variable heavy chain and variable light chainsequences from the starting antibody are analyzed and a human T-cellepitope “map” from each V (variable) region showing the location ofepitopes in relation to complementarity-determining regions (CDRs) andother key residues within the sequence is created. Individual T-cellepitopes from the T-cell epitope map are analyzed in order to identifyalternative amino acid substitutions with a low risk of alteringactivity of the final antibody. A range of alternative variable heavyand variable light sequences are designed comprising combinations ofamino acid substitutions and these sequences are subsequentlyincorporated into a range of binding polypeptides. Typically, between 12and 24 variant antibodies are generated and tested for binding and/orfunction. Complete heavy and light chain genes comprising modifiedvariable and human constant regions are then cloned into expressionvectors and the subsequent plasmids introduced into cell lines for theproduction of whole antibody. The antibodies are then compared inappropriate biochemical and biological assays, and the optimal variantis identified.

The binding specificity and/or affinity of the bispecific antibody toeach target protein can be determined by any conventional assay, forexample, in vitro assays such as immunoprecipitation, radioimmunoassay(RIA), or enzyme-linked immunoabsorbent assay (ELISA), but not belimited thereto.

Alternatively, techniques described for the production of single-chainunits (U.S. Pat. No. 4,694,778, etc.) can be adapted to producesingle-chain units of the present disclosure. Single-chain units areformed by linking the heavy and light chain fragments of the Fv regionvia an amino acid bridge (peptide linker), resulting in a single-chainfusion peptide (scFv). Techniques for the assembly of functional Fvfragments in E. coli may also be used.

Examples of techniques which can be used to produce single-chain Fvs(scFvs) and antibodies include those described in U.S. Pat. Nos.4,946,778, 5,258,498, etc.). For some uses, including in vivo use ofantibodies in humans and in vitro detection assays, it may be preferableto use chimeric, humanized, or human antibodies. A chimeric antibody isa molecule in which different portions of the antibody are derived fromdifferent animal species, such as antibodies having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region. Methods for producing chimeric antibodies are known inthe art. See, e.g., U.S. Pat. Nos. 5,807,715, 4,816,567, and 4, 816,397,which are incorporated herein by reference in their entireties.

Humanized antibodies are antibody molecules derived from a non-humanspecies antibody that bind the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule. Often,framework residues in the human framework regions will be substitutedwith the corresponding residue from the CDR donor antibody to after,preferably improve, antigen-binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen-binding and sequence comparison toidentify unusual framework residues at particular positions (See, e.g.,Queen et al., U.S. Pat. No. 5,585,089, which are incorporated herein byreference in their entireties). Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (U.S. Pat. Nos. 5,225,539, 5,530,101, 5,585,089, etc., eachof which is incorporated by reference in its entirety), veneering orresurfacing (EP 592,106; EP 519,596, each of which is incorporated byreference in its entirety), and chain shuffling (U.S. Pat. No.5,565,332, which is incorporated by reference in its entirety).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods usingantibody libraries derived from human immunoglobulin sequences. Seealso, U.S. Pat. Nos. 4,444,887, 4,716,111, etc., each of which isincorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a desired target polypeptide. Monoclonal antibodies directedagainst the antigen can be obtained from the immunized, transgenic miceusing conventional hybridoma technology. The human immunoglobulintransgenes harbored by the transgenic mice rearrange during B-celldifferentiation, and subsequently undergo class switching and somaticmutation. Thus, using such a technique, it is possible to producetherapeutically useful IgG, IgA, IgM and IgE antibodies.

Completely human antibodies which recognize a selected epitope can alsobe generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope.

In another embodiment, DNA encoding desired monoclonal antibodies may bereadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies). Theisolated and subcloned hybridoma cells serve as a preferred source ofsuch DNA. Once isolated, the DNA may be placed into expression vectors,which are then transfected into prokaryotic or eukaryotic host cellssuch as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO)cells or myeloma cells that do not otherwise produce immunoglobulins.More particularly, the isolated DNA (which may be synthetic as describedherein) may be used to clone constant and variable region sequences forthe manufacture antibodies as described in Newman et al., U.S. Pat. No.5,658,570, which is incorporated by reference herein. Essentially, thisentails extraction of RNA from the selected cells, conversion to cDNA,and amplification by PCR using Ig specific primers. Suitable primers forthis purpose are also described in U.S. Pat. No. 5,658,570. As will bediscussed in more detail below, transformed cells expressing the desiredantibody may be grown up in relatively large quantities to provideclinical and commercial supplies of the immunoglobulin.

Additionally, using routine recombinant DNA techniques, one or more ofthe CDRs of the bispecific antibody may be inserted within frameworkregions, e.g., into human framework regions to humanize a non-humanantibody. The framework regions may be naturally occurring or consensusframework regions, and preferably human framework regions (see, e.g.,Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of humanframework regions). For example, the polynucleotide generated by thecombination of the framework regions and CDRs encodes an antibody thatspecifically binds to at least one epitope of a desired polypeptide,e.g., LIGHT. Preferably, one or more amino acid substitutions may bemade within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen (orepitope). Additionally, such methods may be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentdisclosure and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” by splicing genes from a mouse antibody molecule, ofappropriate antigen specificity, together with genes from a humanantibody molecule of appropriate biological activity can be used. Asused herein, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine monoclonal antibody and a humanimmunoglobulin constant region.

Alternatively, antibody-producing cell lines may be selected andcultured using techniques well known to the skilled artisan. Suchtechniques are described in a variety of laboratory manuals and primarypublications.

Additionally, standard techniques known to those of skill in the art canbe used to introduce mutations in the nucleotide sequence encoding anantibody of the present disclosure, including, but not limited to,site-directed mutagenesis and PCR-mediated mutagenesis which result inamino acid substitutions. Preferably, the variants (includingderivatives) encode less than 50 amino acid substitutions, less than 40amino acid substitutions, less than 30 amino acid substitutions, lessthan 25 amino acid substitutions, less than 20 amino acid substitutions,less than 15 amino acid substitutions, less than 10 amino acidsubstitutions, less than 5 amino acid substitutions, less than 4 aminoacid substitutions, less than 3 amino acid substitutions, or less than 2amino acid substitutions relative to the reference variable heavy chainregion, CDR-H1, CDR-H2, CDR-H3, variable light chain region, CDR-L1,CDR-L2, or CDR-L3. Alternatively, mutations can be introduced randomlyalong all or part of the coding sequence, such as by saturationmutagenesis, and the resultant mutants can be screened for biologicalactivity to identify mutants that retain activity.

Therapeutic Use of the Antibodies

The bispecific antibody provided herein is capable of simultaneouslyblocking the activities of PD-L1 and LAG3, thereby exhibiting improvedeffects in immunotherapies and/or cancer therapies, for example, byactivating immune response (see FIG. 33). Given the ability of thebispecific antibodies of the disclosure to inhibit the binding of LAG-3to MHC Class II molecules and to stimulate antigen-specific T cellresponses, the disclosure also provides a composition or in vitro and invivo methods of using the antibodies of the disclosure to stimulate,enhance or upregulate antigen-specific T cell responses.

An embodiment provides a pharmaceutical composition comprising thebispecific antibody or the anti-PD-L1 or anti-LAG3 antibody as describedabove. The pharmaceutical composition may further comprise apharmaceutically acceptable carrier. The pharmaceutical composition maybe used for stimulating an immune response (e.g., an antigen-specific Tcell response), and/or treating and/or preventing a disease associatedwith PD-L1, LAG3, or both thereof.

Another embodiment provides a method of stimulating an immune response(e.g., an antigen-specific T cell response), and/or treating and/orpreventing a disease associated with PD-L1, LAG3, or both thereof, in asubject in need thereof, comprising administering to the subject apharmaceutically effective amount of the bispecific antibody, theanti-PD-L1 or anti-LAG3 antibody, or the pharmaceutical composition. Themethod may further step of identifying the subject in need of treatingand/or preventing a disease associated with PD-L1, LAG3, or boththereof, prior to the administering step.

The disease associated with PD-L1, LAG3, or both thereof may be selectedfrom cancers (or tumors), infectious diseases, autoimmune reactions,nervous system disorders, and the like.

In an embodiment, the subject may be selected from mammals includinghumans, for example, a mammal (e.g., a human) suffering from a cancerand/or infection mammalian cells. In other embodiment, the subject maybe a cell separated (isolated) from a mammal, for example, a mammalsuffering from the disease selected from cancers infectious diseases,autoimmune reactions, nervous system disorders, and the like (e.g., acancer cell or a cell separated (isolated) from an infectious region inthe mammal, or a T cell, such as a tumor-infiltrating T lymphocyte, aCD4+ T cell, a CD8+ T cell, or the combination thereof).

Another embodiment provides a use of the bispecific antibody, theanti-PD-L1 or anti-LAG3 antibody, or the pharmaceutical composition intreating and/or preventing a cancer or an infection. Another embodimentprovides a use of the bispecific antibody, or the anti-PD-L1 oranti-LAG3 antibody, in preparing a pharmaceutical composition fortreating and/or preventing a cancer or an infection.

In the pharmaceutical compositions, methods and/or uses provided herein,the disease associated with PD-L1, LAG3, or both thereof may be oneassociated with activation (e.g., abnormal activation orover-activation) and/or overproduction (overexpression) of PD-L1, LAG3,or both thereof. For example, the disease may be a cancer or aninfection.

The cancer may be a solid cancer or blood cancer, preferably a solidcancer. The cancer may any tumor expressing PD-L1 protein, and may beselected from the group consisting of bladder cancer, liver cancer,colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma,pancreatic cancer, lung cancer (e.g., small cell lung cancer, non-smallcell lung cancer etc.), breast cancer, urethral cancer, head and neckcancer, gastrointestinal cancer, stomach cancer, oesophageal cancer,ovarian cancer, renal cancer, melanoma, prostate cancer, thyroid cancer,and the like, but may not be limited thereto. In some embodiments, thecancer is selected from the group consisting of bladder cancer, livercancer, pancreatic cancer, non-small cell lung cancer, breast cancer,urethral cancer, colorectal cancer, head and neck cancer, squamous cellcancer, Merkel cell carcinoma, gastrointestinal cancer, stomach cancer,oesophageal cancer, ovarian cancer, renal cancer, small cell lungcancer, and the like. The cancer may be a primary or metastatic cancer.

A specific dosage and treatment regimen for any particular patient willdepend upon a variety of factors, including the particular antibodies,variant or derivative thereof used, the patient's age, body weight,general health, sex, and diet, and the time of administration, rate ofexcretion, drug combination, and the severity of the particular diseasebeing treated. Judgment of such factors by medical caregivers is withinthe ordinary skill in the art. The amount will also depend on theindividual patient to be treated, the route of administration, the typeof formulation, the characteristics of the compound used, the severityof the disease, and the desired effect. The amount used can bedetermined by pharmacological and pharmacokinetic principles well knownin the art.

The administration of the bispecific antibody or the anti-PD-L1 oranti-LAG3 antibody may be conducted through at least one selected fromthe group consisting of intraperitoneal, intravenous, subcutaneous,intradermal, intramuscular, intranasal, epidural, and oral routes, butnot be limited thereto. The bispecific antibody or the anti-PD-L1 oranti-LAG3 antibody or compositions may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Thus, pharmaceutical compositions containingthe antigen-binding polypeptides of the disclosure may be administeredorally, parenterally, intracistemally, intravaginally,intraperitoneally, rectally, topically (as by powders, ointments, dropsor transdermal patch), bucally, or as an oral or nasal spray.

The term “parenteral” as used herein refers to modes of administrationwhich include intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intra-articular injection and infusion.

Administration can be systemic or local. In addition, it may bedesirable to introduce the antibodies of the disclosure into the centralnervous system by any suitable route, including intraventricular andintrathecal injection; intraventricular injection may be facilitated byan intraventricular catheter, for example, attached to a reservoir, suchas an Ommaya reservoir. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent.

It may be desirable to administer the bispecific antibodies, or theanti-PD-L1 or anti-LAG3 antibodies, or compositions of the disclosurelocally to the area in need of treatment; this may be achieved by, forexample, and not by way of limitation, local infusion during surgery,topical application, e.g., in conjunction, with a wound dressing aftersurgery, by injection, by means of a catheter, by means of asuppository, or by means of an implant, said implant being of a porous,non-porous, or gelatinous material, including membranes, such assialastic membranes, or fibers. Preferably, when administering aprotein, including an antibody, of the disclosure, care must be taken touse materials to which the protein does not absorb.

In another embodiment, the bispecific antibodies or the anti-PD-L1 oranti-LAG3 antibodies or composition can be delivered in a vesicle, inparticular a liposome. In yet another embodiment, the bispecificantibodies or the anti-PD-L1 or anti-LAG3 antibodies or composition canbe delivered in a controlled release system. In one embodiment, for thecontrolled release system, any pharmaceutically acceptable pumps, and/orpolymeric materials may be used.

The pharmaceutically effective amount of the bispecific antibodies orthe anti-PD-L1 or anti-LAG3 antibodies for treating, inhibiting,ameliorating, and/or preventing an inflammatory, immune or malignantdisease, disorder, or condition, can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease, disorder or condition, and should be decidedaccording to the judgment of the practitioner and each patient'scircumstances. Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

The methods of treating an infectious or malignant disease (e.g.,cancer), condition or disorder comprising administration of thebispecific antibody or the anti-PD-L1 or anti-LAG3 antibody aretypically tested in vitro, and then in vivo in an acceptable animalmodel, for the desired therapeutic or prophylactic activity, prior touse in humans. Suitable animal models, including transgenic animals, arewell known to those of ordinary skill in the art. For example, in vitroassays to demonstrate the therapeutic utility of the bispecific antibodyor the anti-PD-L1 or anti-LAG3 antibody include the effect of thebispecific antibody or the anti-PD-L1 or anti-LAG3 antibody on a cellline or a patient tissue sample. The effect of the bispecific antibodyor the anti-PD-L1 or anti-LAG3 antibody on the cell line and/or tissuesample can be determined utilizing techniques known to those of skill inthe art, such as the assays disclosed elsewhere herein. In accordancewith the disclosure, in vitro assays which can be used to determinewhether administration of the bispecific antibody or the anti-PD-L1 oranti-LAG3 antibody is indicated, include in vitro cell culture assays inwhich a patient tissue sample is grown in culture, and exposed to orotherwise administered a compound, and the effect of such compound uponthe tissue sample is observed.

Various delivery systems are known and can be used to administer anantibody of the disclosure or a polynucleotide encoding an antibody ofthe disclosure, e.g., encapsulation in liposomes, microparticles,microcapsules, recombinant cells capable of expressing the compound,receptor-mediated endocytosis, construction of a nucleic acid as part ofa retroviral or other vector, etc.

The pharmaceutical compositions may comprise an effective amount of thebispecific antibody or the anti-PD-L1 or anti-LAG3 antibody, and anacceptable carrier. In some embodiments, the composition furtherincludes a second anticancer agent (e.g., an immune checkpointinhibitor).

In a specific embodiment, the term “pharmaceutically acceptable” mayrefer to approved by a regulatory agency of the Federal or a stategovernment or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. Further, a “pharmaceutically acceptable carrier” will generallybe a non-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type.

The term “carrier” may refer to a diluent, adjuvant, excipient, orvehicle with which the therapeutic is administered. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents such as acetates,citrates or phosphates. Antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; and agents forthe adjustment of tonicity such as sodium chloride or dextrose are alsoenvisioned. These compositions can take the form of solutions,suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences by E. W. Martin, incorporated herein byreference. Such compositions will contain a therapeutically effectiveamount of the antigen-binding polypeptide, preferably in purified form,together with a suitable amount of carrier so as to provide the form forproper administration to the patient. The formulation should suit themode of administration. The parental preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

In an embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the disclosure can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

Diagnostic Use of the Antibody

Over-expression and/or over-activation of PD-L1 and/or LAG3 is observedin a biological sample (e.g., cells, tissues, blood, serum, etc.) from apatient suffering from a certain cancer and/or infection (for example,tumor cell or tissue, blood or serum from an infectious patient), and/orpatients having PD-L1- and/or LAG3-over-expressing cells are likelyresponsive to treatments with the bispecific antibody or the anti-PD-L1or anti-LAG3 antibody. Accordingly, the bispecific antibody or theanti-PD-L1 or anti-LAG3 antibody of the present disclosure can also beused for diagnostic and prognostic purposes.

An embodiment provides a pharmaceutical composition for diagnosing adisease associated with PD-L1, LAG3, or both thereof, the compositioncomprising the bispecific antibody or the anti-PD-L1 or anti-LAG3antibody. In another embodiment, provided is a use of the bispecificantibody or the anti-PD-L1 or anti-LAG3 antibody for diagnosing adisease associated with PD-L1, LAG3, or both thereof.

Another embodiment provides a method of diagnosing a disease associatedwith PD-L1, LAG3, or both thereof, the method comprising contacting abiological sample obtained from a patient with the bispecific antibodyor the anti-PD-L1 or anti-LAG3 antibody, and detecting antigen-antibodyreaction or measuring a level of antigen-antibody reaction in thebiological sample. In this method, when the antigen-antibody reaction isdetected in the biological sample or the level of the antigen-antibodyreaction in the biological sample is higher than that of a normalsample, the patient from whom the biological sample is obtained may bedetermined as a patient with a disease associated with PD-L1, LAG3, orboth thereof. Therefore, in some embodiments, the method may furthercomprise contacting a normal sample with the bispecific antibody or theanti-PD-L1 or anti-LAG3 antibody, and measuring a level of anantigen-antibody reaction in the normal sample. In addition, the methodmay further comprise comparing the level of the antigen-antibodyreaction in the biological sample and in the normal sample, after themeasuring step. In addition, after the detecting step or comparing step,the method may further comprise determining the patient as a patientwith a disease associated with PD-L1, LAG3, or both thereof, when theantigen-antibody reaction is detected in the biological sample or thelevel of the antigen-antibody reaction in the biological sample ishigher than that of the normal sample.

The disease associated with PD-L1, LAG3, or both thereof may be oneassociated with activation (e.g., abnormal activation orover-activation) and/or overproduction (overexpression) of PD-1, LAG3,or both thereof. For example, the disease may be a cancer or aninfection, as described above.

In the diagnosing composition and method, the biological sample may beat least one selected from the group consisting of a cell, a tissue,body fluid (e.g., blood, serum, lymph, etc.) and the like, obtained(separated) from a patient to be diagnosed. The normal sample may be atleast one selected from the group consisting of a cell, a tissue, bodyfluid (e.g., blood, serum, lymph, urine, etc.) and the like, obtained(separated) from a patient having no disease associated with PD-L1,LAG3, or both thereof. The patient may be selected from a mammal, suchas a human. Upon optional pre-treatment of the sample, the sample can beincubated with the bispecific antibody or the anti-PD-L1 or anti-LAG3antibody of the present disclosure under conditions allowing theantibody to interact with a PD-L1 and/or LAG3 protein potentiallypresent in the sample.

Presence and/or level (concentration) of the PD-L1 and/or LAG3 proteinin the sample can be used for identifying a patient who is suitable fora treatment with the bispecific antibody or the anti-PD-L1 or anti-LAG3antibody, or a patient who is responsive or susceptive to the treatmentwith the bispecific antibody or the anti-PD-L1 or anti-LAG3 antibody.

An embodiment provides a pharmaceutical composition identifying apatient who is suitable for a treatment with the bispecific antibody orthe anti-PD-L1 or anti-LAG3 antibody, or a patient who is responsive orsusceptive to the treatment with the bispecific antibody or theanti-PD-L1 or anti-LAG3 antibody, the composition comprising thebispecific antibody or the anti-PD-L1 or anti-LAG3 antibody. In anotherembodiment, provided is a use of the bispecific antibody or theanti-PD-L1 or anti-LAG3 antibody for identifying a patient who issuitable for a treatment with the bispecific antibody or the anti-PD-L1or anti-LAG3 antibody, or a patient who is responsive or susceptive tothe treatment with the bispecific antibody or the anti-PD-L1 oranti-LAG3 antibody. Another embodiment provides a method of identifyinga patient who is suitable for a treatment with the bispecific antibodyor the anti-PD-L1 or anti-LAG3 antibody, or a patient who is responsiveor susceptive to the treatment with the bispecific antibody or theanti-PD-L1 or anti-LAG3 antibody, the method comprising contacting abiological sample obtained from a patient with the bispecific antibodyor the anti-PD-L1 or anti-LAG3 antibody, and detecting antigen-antibodyreaction or measuring a level of antigen-antibody reaction in thebiological sample.

An embodiment provides a composition for detection of PD-L1, LAG3, orboth thereof simultaneously, in a biological sample, the compositioncomprising the bispecific antibody or the anti-PD-L1 or anti-LAG3antibody. Another embodiment provides a method of detection of PD-L1,LAG3, or both thereof simultaneously, in a biological sample, the methodcomprising contacting the biological sample with the bispecific antibodyor the anti-PD-L1 or anti-LAG3 antibody; and detecting (measuring) anantigen-antibody reaction (binding) between the bispecific antibody orthe anti-PD-L1 or anti-LAG3 antibody and PD-L1, LAG3, or both thereof.

In the detecting composition and the detecting method, the term“detection of PD-L1, LAG3, or both thereof” may refer to, but not belimited to, detection of presence (and/or absence) and/or level ofPD-L1, LAG3, or both thereof in the biological sample.

In the method of detection, when an antigen-antibody reaction isdetected, it can be determined that PD-L1, LAG3, or both thereof arepresent in the biological sample, and when an antigen-antibody reactionis not detected, it can be determined that PD-L1, LAG3, or both thereofare absent (not present) in the biological sample. Therefore, the methodof detection may further comprise, after the detecting step, determiningthat PD-L1, LAG3, or both thereof are present in the biological samplewhen an antigen-antibody reaction is detected, and/or that PD-L1, LAG3,or both thereof are absent (not present) in the biological sample, whenan antigen-antibody reaction is not detected.

In the method of detection, the level of PD-L1, LAG3, or both thereofmay be determined according to the degree of the antigen-antibodyreaction (e.g., the amount of antigen-antibody complex formed by theantigen-antibody reaction, the intensity of any signal obtained by theantigen-antibody reaction, and the like, which can be measured by anyconventional means).

The biological sample may comprise at least one selected from the groupconsisting of a cell (e.g., a tumor cell), a tissue (e.g., a tumortissue), body fluid (e.g., blood, serum, etc.), and the like, obtainedor isolated from a mammal such as a human. The steps of the method ofdetection may be conducted in vitro.

In the diagnosing method and/or detecting method, the step of detectingthe antigen-antibody reaction or measuring a level of theantigen-antibody reaction may be performed by any general method knownto the relevant art, such as general enzymatic reactions, fluorescentreactions, luminescent reactions, and/or detection of radiation. Forexample, the step may be performed by a method selected from, but notlimited to, the group consisting of immunochromatography,immunohistochemistry (IHC), enzyme linked immunosorbent assay (ELISA),radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescenceimmunoassay (FIA), luminescence immunoassay (LIA), western blotting,microarray, flow cytometry, surface plasmon resonance (SPR), and thelike, but not be limited thereto.

Polynucleotides Encoding the Antibodies and Methods of Preparing theAntibodies

An embodiment provides a polynucleotide encoding the bispecific antibodyor the anti-PD-L1 or anti-LAG3 antibody. In particular, an embodimentprovides a polynucleotide encoding a heavy chain of the bispecificantibody in an IgG-scFv form. Other embodiment provides a polynucleotideencoding a light chain of the bispecific antibody in the IgG-scFv form.The IgG-scFv form may refer to a kind of a bispecific antibodycomprising a full-length IgG antibody targeting (binding to) one ofPD-L1 and LAG3 proteins and a scFv fragment targeting (binding to) theother one, wherein the scFv is linked to a C-terminus and/or N-terminusof the full-length IgG antibody directly (without a peptide linker) orvia a peptide linker.

In an embodiment, when the bispecific antibody in an IgG-scFv formcomprises a full-length IgG antibody against PD-L1 and a scFv fragmentagainst LAG3, the polynucleotide encoding a heavy chain of thebispecific antibody may encode a heavy chain of the full-length IgGantibody against PD-L1 and a scFv fragment against LAG3 that is linkedto a C-terminus and/or N-terminus of the full-length IgG antibodydirectly or via a peptide linker; and the polynucleotide encoding alight chain of the bispecific antibody may encode a light chain of thefull-length IgG antibody against PD-1.

In another embodiment, when the bispecific antibody in an IgG-scFv formcomprises a full-length IgG antibody against LAG3 and a scFv fragmentagainst PD-L1, the polynucleotide encoding a heavy chain of thebispecific antibody may encode a heavy chain of the full-length IgGantibody against LAG3 and a scFv fragment against PD-L1 that is linkedto a C-terminus and/or N-terminus of the full-length IgG antibodydirectly or via a peptide linker; and the polynucleotide encoding alight chain of the bispecific antibody may encode a light chain of thefull-length IgG antibody against LAG3.

Another embodiment provides a recombinant vector comprising thepolynucleotide encoding a heavy chain of the bispecific antibody, thepolynucleotide encoding a light chain of the bispecific antibody, orboth thereof. Another embodiment provides a recombinant cell transfectedwith the recombinant vector.

Another embodiment provides a method of preparing the bispecificantibody, comprising expressing the polynucleotide encoding a heavychain of the bispecific antibody, the polynucleotide encoding a lightchain of the bispecific antibody in a cell. The step of expressing thepolynucleotide may be conducted by culturing the cell comprising thepolynucleotide (for example, in a recombinant vector) under a conditionallowing the expression of the polynucleotide. The method may furthercomprise isolating and/or purifying the bispecific antibody from thecell culture, after the step of expressing or culturing.

EXAMPLES

Hereafter, the present invention will be described in detail byexamples.

The following examples are intended merely to illustrate the inventionand are not construed to restrict the invention.

Example 1: Preparation of Anti-PD-L1 Monoclonal Antibodies

1.1. Preparation of Anti-Human-PD-L1 Mouse Monoclonal Antibodies andAnalysis Thereof

Anti-human-PD-L1 mouse monoclonal antibodies were generated using thehybridoma technology.

Antigen: human PD-L1-Fc protein and human PD-L1 highly expressed CHOK1cell line (PDL1-CHOK1 cell line).

Immunization: To generate mouse monoclonal antibodies to human PD-L1,6-8 week female BALB/c mice were firstly immunized with 1.5×10⁷PDL1-CHOK1 cells. Day 14 and 33 post first immunization, the immunizedmice were re-immunized with 1.5×10⁷ PDL1-CHOK1 cells respectively. Toselect mice producing antibodies that bound PD-L1 protein, sera fromimmunized mice were tested by ELISA. Briefly, microtiter plates werecoated with human PD-L1 protein at 1 μg/ml in PBS, 100 μl/well at roomtemperature (RT) overnight, then blocked with 100 μl/well of 5% BSA.Dilutions of plasma from immunized mice were added to each well andincubated for 1-2 hours at RT. The plates were washed with PBS/Tween andthen incubate with anti-mouse IgG antibody conjugated with Horse RadishPeroxidase (HRP) for 1 hour at RT. After washing, the plates weredeveloped with ABTS substrate and analyzed by spectrophotometer at OD405 nm. Mice with sufficient titers of anti-PDL1 IgG were boosted with50 μg human PDL1-Fc protein at Day 54 post-immunization. The resultingmice were used for fusions. The hybridoma supernatants were tested foranti-PD-L1 IgGs by ELISA.

The amino acid and polynucleotide sequences of the variable regions ofHybridoma HL1210-3 are provided in Table 5 below.

TABLE 5 HL1210-3 variable sequences Name Sequence SEQ ID NO: HL1210-3 VHGAAGTGAAACTGGTGGAGTCTGGGGGAGACTTAGTGAAGC 112CTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGACATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGTCTGGAGTGGGTCGCAACCATTAGTGATGGTGGTGGTTACATCTACTATTCAGACAGTGTGAAGGGGCGATTTAGCATCTCCAGAGACAATGCCAAGAACAACCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCTTGTATATTTGTGCAAGAGAATTTGGTAAGCGCTATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA HL1210-3 VHEVKLVESGGDLVKPGGSLKLSCAASGFTFSSYDMSWVRQT 113PEKSLEWVATISDGGGYIYYSDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYICAREFGKRYALDYWGQGTSVT HL1210-3 VLGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACAT 114CGGTAGGAGACAGGGTCAGCATCTCCTGCAAGGCCAGTCAGGATGTGACTCCTGCTGTCGCCTGGTATCAACAGAAGCCAGGACAATCTCCTAAACTACTGATTTACTCCACATCCTCCCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATACTACTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAA A HL1210-3 VLDIVMTQSHKFMSTSVGDRVSISCKASQDVTPAVAWYQQKP 115GQSPKLLIYSTSSRYTGVPDRFTGSGSGTDFTFTISSVQA EDLAVYYCQQHYTTPLTFGAGTKLELK

1.2. Activities of HL1210-3 Mouse mAb

To evaluate the binding activity of hybridoma clone HL1210-3, thepurified mAb from this clone were subjected to ELISA test. Briefly,microtiter plates were coated with human PD-L1-Fc protein at 0.1 μg/mlin PBS, 100 μl/well at 4° C. overnight, then blocked with 100 μl/well of5% BSA. Three-fold dilutions of HL1210-3 antibodies starting from 0.2μg/ml were added to each well and incubated for 1-2 hours at RT. Theplates were washed with PBS/Tween and then incubate with goat-anti-mouseIgG antibody conjugated with Horse Radish Peroxidase (HRP) for 1 hour atRT. After washing, the plates were developed with TMB substrate andanalyzed by spectrophotometer at OD 450-630 nm. As shown in FIG. 1,HL1210-3 can bind to human PD-L1 with high activity (EC₅₀=5.539 ng/ml).

To evaluate the activity of HL1210-3 mouse mAb to block human PD-L1binding to its receptor PD-1, a receptor blocking assay was performed byusing recombinant human PD-1.

To evaluate the blocking effect of HL1210-3 mouse mAb on recombinanthuman PD-L1 to bind to its receptor PD-1, the ELISA based receptorblocking assay was employed. Briefly, microtiter plates were coated withhuman PD-L1-Fc protein at 1 μg/ml in PBS, 100 μl/well at 4° C.overnight, then blocked with 100 μl/well of 5% BSA. 50 μl biotin-labeledhuman PD-1-Fc protein and 3-fold dilutions of HL1210-3 antibodiesstarting from 2 μg/ml at 50 μl were added to each well and incubated for1 hour at 37° C. The plates were washed with PBS/Tween and thenincubated with Streptavidin-HRP for 1 hour at 37° C. After washing, theplates were developed with TMB substrate and analyzed byspectrophotometer at OD 450-630 nm. As shown in FIG. 2, HL1210-3 canefficiently inhibit the binding of human PD-L1 to human PD1 atIC₅₀=0.7835 nM.

In addition, a receptor blocking assay was also performed by usingmammalian cell expressed human PD-1.

To evaluate the blocking effect of HL1210-3 mouse mAb on human PD-L1expressed on mammalian cells to bind to its receptor PD-1, theFACS-based receptor blocking assay was used. Briefly, PDL1-CHOK1 cellswere firstly incubated with 3-fold serious diluted HL1210-3 mouse mAbstarting at 20 μg/ml at RT for 1 hour. After wash by FACS buffer (PBSwith 2% FBS), the biotin-labeled huPD-1 was added to each well andincubated at RT for 1 hour. Then, the Streptavidin-PE was added to eachwell for 0.5 hour post twice wash with FACS buffer. The mean florescenceintensity (MFI) of PE was evaluated by FACSAriaIII. As shown in FIG. 3,the HL1210-3 antibody can highly efficiently inhibit the binding of PD-1on PD-L1 expressed on mammalian cells at IC50 of 2.56 nM with 92.6% topinhibition rate.

${\%\mspace{14mu}{of}\mspace{14mu}{inhibition}} = {\left( {1 - \frac{{MFI}\mspace{14mu}{of}\mspace{14mu}{testing}\mspace{14mu}{antibody}}{{MFI}\mspace{14mu}{of}\mspace{14mu}{vehicle}\mspace{14mu}{contori}}} \right) \times 100\%}$

1.3. Effects of HL1210-3 Mouse mAb

To evaluate the effect of HL1210-3 mouse mAb to promote human T cellimmune response, the response of human T cells assessed in a mixedlymphocyte reaction setting. Human DCs were differentiated fromCD14+monocytes in the presence of GM-CSF and IL-4 for 7 days. CD4+ Tcells isolated from another donor were then co-cultured with the DCs andseral dilutions of anti-PD-L1 blocking antibody. At day 5post-inoculation, the culture supernatant was assayed for IFNγproduction. The results indicated that the HL1210-3 antibodies candose-dependently promote IFNγ production, suggesting anti-PD-L1 antibodycan promote human T cell response (FIG. 4).

1.4. Binding Affinity of HL1210-3 Mouse mAb

The binding of the HL1210-3 antibodies to recombinant PD-L1 protein(human PD-L1-his taq) was tested with BIACORE™ using a capture method.The HL1210-3 mouse mAb was captured using anti-mouse Fc antibody coatedon a CM5 chip. A series dilution of human PD-L1-his taq protein wasinjected over captured antibody for 3 mins at a flow rate of 25 μg/ml.The antigen was allowed to dissociate for 900 s. All the experiment werecarded out on a Biacore T200. Data analysis was carried out usingBiacore T200 evaluation software. The results are shown in FIG. 5 andTable 6 below.

TABLE 6 Binding Kinetics of HL1210-3 to recombinant human PD-L1 Antibodyka (1/Ms) kd (1/s) KD (M) HL1210-3 1.61E+05 4.69E−05 2.93E−10

1.5. Humanization of the HL1210-3 Mouse mAb

The mAb HL1210-3 variable region genes were employed to create ahumanized MAb. In the first step of this process, the amino acidsequences of the VH and VK of MAb HL1210-3 were compared against theavailable database of human Ig gene (IgG1) sequences to find the overallbest-matching human germline Ig gene sequences. For the light chain, theclosest human match was the O18/Jk2 and KV1-39*01/KJ2*04 gene, and forthe heavy chain the closest human match was the VH3-21 gene. VH3-11,VH3-23, VH3-7*01 and VH3-48 genes were also selected due to their closematches.

Humanized variable domain sequences were then designed where the CDR1(SEQ ID NO. 4), 2 (SEQ ID NO. 5) and 3 (SEQ ID NO. 6) of the HL1210-3light chain were grafted onto framework sequences of the O18/Jk2 andKV1-39*01/KJ2*04 gene, and the CDR1 (SEQ ID NO. 1), 2 (SEQ ID NO. 2),and 3 (SEQ ID NO. 3) sequences of the HL1210-3 VH were grafted ontoframework sequences of the VH3-21, VH3-11, VH3-23, VH3-48 or VH3-7*01gene. A 3D model was then generated to determine if there were anyframework positions where replacing the mouse amino acid to the humanamino acid could affect binding and/or CDR conformation. In the case ofthe light chain, 22S, 43S, 60D, 63T and 42Q (Kabat numbering, see Table7) in framework were identified. In the case of the heavy chain, 1E,37V, 40T, 44S, 49A, 77N, 911, 94R and 108T in the framework was involvedin back-mutations.

TABLE 7 Humanization Design Construct Mutation VH Design I: VH3-21/JH6Hu1210 VH Chimera Hu1210 VH.1 CDR-grafted Hu1210 VH.1a S49A Hu1210 VH.1bS49A, G44S, Y91I VH Design II: VH3-11/JH6 Hu1210 VH.2 CDR-grafted, Q1EHu1210 VH.2a Q1E, S49A Hu1210 VH.2b Q1E, I37V, S49A, G44S, Y91I VHDesign III: VH3-23/JH6 Hu1210 VH.3 CDR-grafted, K94R Hu1210 VH.3a G44S,S49A, Y91I, K94R VH Design IV: VH3-48/JH6 Hu1210 VH.4 CDR-grafted Hu1210VH.4a S49A Hu1210 VH.4b S49A, G44S, Y91I Hu1210 VH.4c D52E, S49A, G44S,Y91I Hu1210 VH.4d G53A, S49A, G44S, Y91I Hu1210 VH.4e G53V, S49A, G44S,Y91I VH Design V: VH3-7*01/HJ1*01 Hu1210 VH.5 CDR-grafted Hu1210 VH.5aH91I Hu1210 VH.5b H91I, H108T Hu1210 VH.5c H91I, H77N Hu1210 VH.5d H91I,H77N, H40T VK Design I: 018/Jk2 Hu1210 Vk Chimera Hu1210 Vk.1CDR-grafted Hu1210 Vk.1a A43S VK Design II: KV1-39*01/KJ2*04 Hu1210 Vk.2CDR-grafted Hu1210 Vk.2a L60D, L63T Hu1210 Vk.2b L60D, L63T, L42Q, L43SHu1210 Vk.2c L60D, L63T, L42Q, L43S, T22S

The amino acid and nucleotide sequences of some of the humanizedantibody are listed in Table 8 below.

TABLE 8 Humanized antibody sequences (bold indicates CDR) NameAmino Acid Sequence SEQ ID NO: HL1210-VHEVKLVESGGDLVKPGGSLKLSCAASGFTFSSYDMSWVRQTPEKSLEWVATIS 7DGGGYIYYSDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYICAREFGKRY ALDYWGQGTSVTVSSHu1210 VH.1 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTIS 8DGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREFGKRY ALDYWGQGTTVTVSSHu1210 VH.1a EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATIS 9DGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREFGKRY ALDYWGQGTTVTVSSHu1210 VH.1b EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVATIS 10DGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYICAREFGKRY ALDYWGQGTTVTVSSHu1210 VH.2 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWIRQAPGKGLEWVSTIS 11DGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREFGKRY ALDYWGQGTTVTVSSHu1210 VH.2a EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWIRQAPGKGLEWVATIS 12DGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREFGKRY ALDYWGQGTTVTVSSHu1210 VH.2b EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVATIS 13DGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYICAREFGKRY ALDYWGQGTTVTVSSHu1210 VH.3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTIS 14DGGGYIYYSDSVKGRFTTSRDNSKNTLYLQMNSLRAEDTAVYYCAREFGKRY ALDYWGQGTTVTVSSHu1210 VH.3a EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVATIS 15DGGGYIYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYICAREFGKRY ALDYWGQGTTVTVSSHu1210 VH.4 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTIS 16DGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAREFGKRY ALDYWGQGTTVTVSSHu1210 VH.4a EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATIS 17DGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAREFGKRY ALDYWGQGTTVTVSSHu1210 VH.4b EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVATIS 18DGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREFGKRY ALDYWGQGTTVTVSSHu1210 VH.4c EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVATIS 19EGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREFGKRY ALDYWGQGTTVTVSSHu1210 VH.4d EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVATIS 20DAGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREFGKRY ALDYWGQGTTVTVSSHu1210 VH.4e EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVATIS 21DVGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREFGKRY ALDYWGQGTTVTVSSHu1210 VH.5 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATIS 22DGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREFGKRY ALDYWGQGTLVTVSSHU1210 VH.5a EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATIS 23DGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYICAREFGKRY ALDYWGQGTLVTVSSHU1210 VH.5b EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATIS 24DGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYICAREFGKRY ALDYWGQGTTVTVSSHU1210 VH.5C EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATIS 25DGGGYIYYSDSVKGRFTISRDNAKNNLYLQMNSLRAEDTAVYICAREFGKRY ALDYWGQGTLVTVSSHU1210 VH.5d EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQTPEKSLEWVATIS 26DGGGYIYYSDSVKGRFTISRDNAKNNLYLQMNSLRAEDTAVYICAREFGKRY ALDYWGQGTLVTVSSHL1210-VK DIVMTQSHKFMSTSVGDRVSISCKASQDVTPAVAWYQQKPGQSPKLLIYSTS 27SRYTGVPDRFTGSGSGTDFTETISSVQAEDLAVYYCQQHYTTPLTFGAGTKL ELK Hu1210 VK.1DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYSTS 28SRYTGVPSRFSGSGSGTDFTETTSSLQPEDIATYYCQQHYTTPLTFGQGTKL EIK Hu1210 VK.1aDIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKSPKLLIYSTS 29SRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQGTKL EIK Hu1210 Vk.2DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYSTS 30SRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPLTFGQGTKL EIKR Hu1210 Vk.2aDIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYSTS 31SRYTGVPDRFTGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPLTFGQGTKL EIKR Hu1210 Vk.2bDIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGQSPKLLIYSTS 32SRYTGVPDRFTGSGSGTDFTLTISSLOPEDFATYYCQQHYTTPLTFGQGTKL EIKR Hu1210 Vk.2cDIQMTOSPSSLSASVGDRVTISCKASQDVTPAVAWYQQKPGQSPKLLIYSTS 33SRYTGVPDRFTGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPLTFGQGTKL EIKR NameNucleic Acid Sequence SEQ ID NO: HL1210 VHGAGGTGAAGCTGGTGGAGAGCGGCGGAGATCTGGTGAAGCCTGGCGGCAGCC 34TGAAGCTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGGCAGACCCCCGAGAAGAGCCTGGAGTGGGTGGCCACCATCAGCGATGGCGGCGGCTACATCTACTACAGCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAACCTGTACCTGCAGATGAGCAGCCTGAGGAGCGAGGACACCGCCCTGTACATCTGCGCCAGGGAGTTCGGCAAGAGGTACGCCCTGGACTACTGGGGACAGGGCACCAGCGTGACCGTGAGCAGC Hu1210 VH.1GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCC 35TGAGACTGAGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGAGCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.1aGAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCC 36TGAGACTGAGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.1bGAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCC 37TGAGACTGAGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTCGGCAAATGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.2GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCC 38TGAGACTGAGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGAGCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.2aGAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCC 39TGAGACTGAGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.2bGAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCC 40TGAGACTGAGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.3GAGGTGCAGCTGCTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCC 41TGAGACTGAGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGAGCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGG Hu1210 VH.3aGAGGTGCAGCTGCTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCC 42TGAGACTGAGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTAGATCTATTAGTCCGAGAGCGTGAAGGGGAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.4GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCC 43TGAGACTGAGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGAGCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.4aGAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCC 44TGAGACTGAGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTAGATCTATTAGTCCGAGAGCGTGAAGGGCAGGTTCACCATCAGGAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.4bGAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCC 45TGAGACTGAGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.4cGAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCC 46TGAGACTGAGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGAAGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210_VH.4dGAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCC 47TGAGACTGAGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGCGGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210_VH.4eGAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCC 48TGAGACTGAGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGTTGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.5GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCC 49TGAGGCTGTCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTACTCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTATTACTGCGCCAGGGAGTTTGGCAAGAGGTACGCCCTGGATTACTGGGGCCAGGGCACACTGGTGACAGTGAGCTCC Hu1210 VH.5aGAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCC 50TGAGGCTGTCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTACTCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTATATCTGCGCCAGGGAGTTTGGCAAGAGGTACGCCCTGGATTACTGGGGCCAGGGCACACTGGTGACAGTGAGCTCC Hu1210 VH.5bGAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCC 51TGAGGCTGTCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTACTCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACAACCTGTACCTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTATATCTGCGCCAGGGAGTTTGGCAAGAGGTACGCCCTGGATTACTGGGGCCAGGGCACACTGGTGACAGTGAGCTCC Hu1210 VH.5cGAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCC 52TGAGGCTGTCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGGTGAGGCAGACCCCTGAGAAGAGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTACTCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACAACCTGTACCTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTATATCTGCGCCAGGGAGTTTGGCAAGAGGTACGCCCTGGATTACTGGGGCCAGGGCACACTGGTGACAGTGAGCTCC Hu1210_VH.5dGAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCC 53TGAGGCTGTCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTACTCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTATATCTGCGCCAGGGAGTTTGGCAAGAGGTACGCCCTGGATTACTGGGGCCAGGGCACAACCGTGACAGTGAGCTCC HL1210 VKGACATCGTGATGACCCAGAGCCACAAGTTCATGAGCACCAGCGTGGGCGATA 54GGGTGAGCATCAGCTGCAAGGCCAGCCAGGATGTGACCCCTGCCGTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAGCACCAGCAGCAGGTACACCGGCGTGCCCGACAGGTTCACAGGAAGCGGCAGCGGCACCGACTTCACCTTCACCATCAGCAGCGTGCAGGCCGAGGACCTGGCCGTGTACTACTGCCAGCAGCACTACACCACCCCTCTGACCTTCGGCGCCGGCACCAAGCTG GAGCTGAAGHu1210 VK.1 GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACA 55GGGTGACCATCACCTGCAAGGCCAGCCAGGATGTGACCCCTGCCGTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAGCACCAGCAGCAGGTACACCGGCGTGCCCAGCAGGTTTAGCGGAAGCGGCAGCGGCACCGACTTCACCTTCACCATCAGCAGCCTGCAGCCCGAGGACATCGCCACCTACTACTGCCAGCAGCACTACACCACCCCTCTGACCTTCGGCCAGGGCACCAAGCTG GAGATCAAGHu1210 VK.1a GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACA 56GGGTGACCATCACCTGCAAGGCCAGCCAGGATGTGACCCCTGCCGTGGCCTGGTACCAGCAGAAGCCCGGCAAGTCCCCCAAGCTGCTGATCTACAGCACCAGCAGCAGGTACACCGGCGTGCCCAGCAGGTTTAGCGGAAGCGGCAGCGGCACCGACTTCACCTTCACCATCAGCAGCCTGCAGCCCGAGGACATCGCCACCTACTACTGCCAGCAGCACTACACCACCCCTCTGACCTTCGGCCAGGGCACCAAGCTG GAGATCAAGHu1210 VK.2 GACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCCGCTTCCGTGGGCGACA 57GGGTGACCATCACCTGCAAGGCCAGCCAGGACGTGACACCTGCTGTGGCCTGGTATCAACAGAAGCCTGGCAAGGCTCCTAAGCTCCTGATCTACAGCACATCCTCCCGGTACACCGGAGTGCCCTCCAGGTTTAGCGGCAGCGGCTCCGGCACCGATTTCACCCTGACCATTTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACACCCCTGACCTTCGGCCAGGGCACCAAGCTG GAGATCAAGCGGHu1210 VK.2a GACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCCGCTTCCGTGGGCGACA 58GGGTGACCATCACCTGCAAGGCCAGCCAGGACGTGACACCTGCTGTGGCCTGGTATCAACAGAAGCCTGGCAAGGCTCCTAAGCTCCTGATCTACAGCACATCCTCCCGGTACACCGGAGTGCCCGACAGGTTTACCGGCAGCGGCTCCGGCACCGATTTCACCCTGACCATTTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACACCCCTGACCTTCGGCCAGGGCACCAAGCTG GAGATCAAGCGGHu1210 VK.2b GACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCCGCTTCCGTGGGCGACA 59GGGTGACCATCACCTGCAAGGCCAGCCAGGACGTGACACCTGCTGTGGCCTGGTATCAACAGAAGCCTGGCCAGAGCCCTAAGCTCCTGATCTACAGCACATCCTCCCGGTACACCGGAGTGCCCGACAGGTTTACCGGCAGCGGCTCCGGCACCGATTTCACCCTGACCATTTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACACCCCTGACCTTCGGCCAGGGCACCAAGCTG GAGATCAAGCGGHu1210 VK.2c GACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCCGCTTCCGTGGGCGACA 60GGGTGACCATCAGCTGCAAGGCCAGCCAGGACGTGACACCTGCTGTGGCCTGGTATCAACAGAAGCCTGGCCAGAGCCCTAAGCTCCTGATCTACAGCACATCCTCCCGGTACACCGGAGTGCCCGACAGGTTTACCGGCAGCGGCTCCGGCACCGATTTCACCCTGACCATTTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACACCCCTGACCTTCGGCCAGGGCACCAAGCTG GAGATCAAGCGG

The humanized VH and VK genes were produced synthetically and thenrespectively cloned into vectors containing the human gamma 1 and humankappa constant domains. The pairing of the human VH and the human VKcreated the 40 humanized antibodies (see Table 9).

TABLE 9 Humanized antibodies with their VH an VL regions VH Vk Hu1210VH.1 Hu1210 VH.1a Hu1210 VH.1b Hu1210 VH.2 Hu1210 VH.2a Hu1210 VH 2.bHu1210 VH Hu1210 Vk.1 Hu1210-1 Hu1210-2 Hu1210-3 Hu1210-4 Hu1210-5Hu1210 Vk.1a Hu1210-7 Hu1210-8 Hu1210-9 Hu1210-10 Hu1210-11 Hu1210 VkH1210 chimera VH Vk Hu1210 VH.3 Hu1210 VH.3a Hu1210 VH.4 Hu1210 VH.4aHu1210 VH.4b Hu1210 Vk.1 Hu1210-13 Hu1210-14 Hu1210-15 Hu1210-16Hu1210-17 Hu1210 Vk.1a Hu1210-18 Hu1210-19 Hu1210-20 Hu1210-21 Hu1210-22VH VK Hu1210 VH.5 HU1210 VH.5a HU1210 VH.5b HU1210 VH.5c HU1210 VH.SdHu1210 Vk.2 Hu1210-23 Hu1210-27 Hu1210-31 Hu1210-32 Hu1210-36 Hu1210Vk.2a Hu1210-24 Hu1210-28 Hu1210-33 Hu1210-37 Hu1210 Vk.2b Hu1210-25Hu1210-29 Hu1210-34 Hu1210-38 Hu1210 Vk.2c Hu1210-26 Hu1210-30 Hu1210-35Hu1210-39 VH Vk Hu1210 VH.4c Hu1210 VH.4d Hu1210 VH.4e Hu1210 Vk.1Hu1210-40 Hu1210-41 Hu1210-42

1.6. Antigen Binding Properties of Humanized PD-L1 Antibodies

To evaluate the antigen binding activity, the humanized antibodies weresubjected to ELISA test. Briefly, microtiter plates were coated withhuman PD-L1-Fc protein at 0.1 μg/ml in PBS, 100 μl/well at 4° C.overnight, then blocked with 100 μl/well of 5% BSA. Five-fold dilutionsof humanized antibodies starting from 10 μg/ml were added to each welland incubated for 1-2 hours at RT. The plates were washed with PBS/Tweenand then incubate with goat-anti-mouse IgG antibody conjugated withHorse Radish Peroxidase (HRP) for 1 hour at RT. After washing, theplates were developed with TMB substrate and analyzed byspectrophotometer at OD 450-630 nm. As shown in FIGS. 6A-6E, all thehumanized antibodies show comparable binding efficacy to human PD-L1 incontact to chimeric antibody.

To evaluate the antigen binding property, the humanized antibodies wereanalyzed for its binding to mammalian expressed PD-L1 by FACS. Briefly,PDL1-CHOK1 cells were firstly incubated with 5-fold serious dilutedhumanized antibodies starting at 2 μg/ml at RT for 1 hour. After wash byFACS buffer (PBS with 2% FBS), the alexa 488-anti-human IgG antibody wasadded to each well and incubated at RT for 1 hour. The MFI of Alexa 488was evaluated by FACSAriaIII. As shown in the FIGS. 7A-7C, all thehumanized antibodies can high efficiently bind to PD-L1 expressed onmammalian cells, which was comparable with chimeric antibody.

To explore the binding kinetics of the humanized antibody, this exampleperformed the affinity ranking by using Octet Red 96. As shown in Table10, hu1210-3, hu1210-8, hu1210-9, hu1210-14, hu1210-17, hu1210-1 andHu1210-22 show better affinity, which is comparable with chimericantibody.

TABLE 10 Affinity ranking of humanized antibodies Kon Antibody KD (M)(1/Ms) kdis(1/s) Hu1210 (mIgG) 7.16E−09 3.94E+05 2.83E−03 H1210 chimera1.07E−09 1.62E+05 1.73E−04 Hu1210-1 4.25E−09 7.10E+04 3.02E−04 Hu1210-23.23E−09 7.78E+04 2.51E−04 Hu1210-3 2.64E−09 8.62E+04 2.28E−04 Hu1210-47.68E−09 7.12E+04 5.46E−04 Hu1210-5 4.83E−09 7.93E+04 3.83E−04 Hu1210-74.78E−09 8.45E+04 4.04E−04 Hu1210-8 1.64E−09 7.72E+04 1.27E−04 Hu1210-92.33E−09 8.37E+04 1.95E−04 Hu1210-10 7.03E−09 8.59E+04 6.04E−04Hu1210-11 4.18E−09 7.54E+04 3.15E−04 Hu1210-13 4.36E−09 8.38E+043.66E−04 Hu1210-14 2.34E−09 8.41E+04 1.97E−04 Hu1210-15 4.45E−097.87E+04 3.50E−04 Hu1210-16 3.14E−09 8.41E+04 2.64E−04 Hu1210-172.20E−09 8.17E+04 1.80E−04 Hu1210-18 4.50E−09 7.92E+04 3.57E−04Hu1210-19 2.50E−09 9.03E+04 2.25E−04 Hu1210-20 4.51E−09 8.87E+044.00E−04 Hu1210-21 3.12E−09 9.39E+04 2.93E−04 Hu1210-22 2.56E−099.00E+04 2.30E−04

The binding of the humanized antibodies to recombinant PD-L1 protein(human PD-L1-his taq) was tested by BIACORE™ using a capture method. TheHL1210-3 mouse mAb were captured using anti-mouse Fc antibody coated ona CM5 chip. A series dilution of human PD-L1-his taq protein wasinjected over captured antibody for 3 mins at a flow rate of 25 μg/ml.The antigen was allowed to dissociate for 900 s. All the experimentswere carried out on a Biacore T200. Data analysis was carried out usingBiacore T200 evaluation software and is shown in Table 11 below.

TABLE 11 Affinity by Biacore Antibody ka (1/Ms) kd (1/s) KD (M) Hu1210-89.346E+4 7.169E−5 7.671E−10 Hu1210-9 9.856E+4 4.528E−5 4.594E−10Hu1210-14 1.216E+5 5.293E−5 4.352E−10 Hu1210-16 9.978E+4 6.704E−56.720E−10 Hu1210-17 1.101E+5 2.128E−5 1.933E−10 Hu1210-28 1.289E+51.080E−4 8.378E−10 Hu1210-31 1.486E+5 1.168E−4 7.862E−10 Hu1210-361.461E+5 7.852E−5 5.376E−10 Hu1210-40  8.77E+04  1.31E−04  1.49E−09Hu1210-41  9.17E+04  3.46E−05  3.78E−10 Hu1210-42  8.68E+04  7.53E−05 8.67E−10 1210 Chimera 1.236E+5 3.265E−5 2.642E−10

1.7. Cross Species Activity

To evaluate the binding of humanized antibodies to huPD-L1, Mouse PD-L1,Rat PD-L1, Rhesus PD-1, the antibodies were performed for the ELISAtesting. Briefly, microtiter plates were coated with human, mouse, ratand rhesus PD-L1-Fc protein at 1 μg/ml in PBS, 100 μl/well at 4° tovernight, then blocked with 100 μg/well of 5% BSA. Three-fold dilutionsof humanized antibodies starting from 1 μg/ml were added to each welland incubated for 1-2 hours at RT. The plates were washed with PBS/Tweenand then incubate with goat-anti-mouse IgG antibody conjugated withHorse Radish Peroxidase (HRP) for 1 hour at RT. After washing, theplates were developed with TMB substrate and analyzed byspectrophotometer at OD 450-630 nm. The Hu1210-41 antibody can bind torhesus PD-L1 with lower affinity and cannot bind to rat and mouse PD-L1(FIG. 8 & Table 12).

TABLE 12 Human Rhesus Rat Mouse EC50 0.215 nM 0.628 nM No binding Nobinding

To evaluate the binding of humanized anti-PD-L1 antibody to human B7family and other immune checkpoint, the antibody was evaluate for itsbinding to B7-H1 (PD-L1), B7-DC, B7-1, B7-2, B7-H2, PD-1, CD28, CTLA4,ICOS and BTLA by ELISA. As shown in FIG. 9, the Hu1210-41 antibody canonly specifically bind to B7-H1 (PD-L1).

1.8. Activity of Humanized Anti-PD-L1 Antibodies to Block Human PD-L1 toPD-1

Cell Based Receptor Blocking Assay

To evaluate the blocking effect of humanized antibodies on human PD-L1expressed on mammalian cells to bind to its receptor PD-1, theFACS-based receptor blocking assay was employed. Briefly, PDL1-CHOK1cells were firstly incubated with 3-fold serious diluted HL1210-3 mousemAb starting at 20 μg/ml at RT for 1 hour. After wash by FACS buffer(PBS with 2% FBS), the biotin-labeled huPD-1 were added to each well andincubated at RT for 1 hour. Then, the Streptavidin-PE was added to eachwell for 0.5 hour post twice wash with FACS buffer. The mean florescenceintensity (MFI) of PE was evaluated by FACSAriaIII.

${\%\mspace{14mu}{of}\mspace{14mu}{inhibition}} = {\left( {1 - \frac{{MFI}\mspace{14mu}{of}\mspace{14mu}{testing}\mspace{14mu}{antibody}}{{MFI}\mspace{14mu}{of}\mspace{14mu}{vehicle}\mspace{14mu}{contori}}} \right) \times 100\%}$

As shown in Table 13 below, Hu1210-3, Hu1210-9, Hu1210-8, Hu1210-14,Hu1210-17, Hu1210-19 and Hu1210-22 antibodies show comparable efficacywith chimeric antibody to blocking the binding of PD-L1 to PD-1.

TABLE 13 PD-1 receptor blocking assay Bio-PD1(30 μg/ml) TOP EC50 H1210chimera 87.16 3.961 Hu1210-8 86.35 4.194 Hu1210-9 85.7 4.038 Hu1210-1688.02 5.436 Hu1210-17 80.88 4.424 Hu1210-3 84.28 3.693 Hu1210-14 79.563.572 Hu1210-19 87.45 4.52 Hu1210-22 85.83 4.505 Hu1210-27 103.9 11.48Hu1210-31 92.91 6.179 Hu1210-36 91.75 8.175

Receptor Blocking Assay by Using Recombinant Human PD-L1

There are two receptors i.e. PD-1 and B7-1 for human PD-L1. To explorethe blocking property of humanized PD-L1 antibody to these two proteins,the protein based receptor blocking assay was employed here. Briefly,microtiter plates were coated with human PD-L1-Fc protein at 1 μg/ml inPBS, 100 μl/well at 4° C. overnight, then blocked with 200 μl/well of 5%BSA at 37° C. for 2 hr. 50 μl biotin-labeled human PD-1-Fc or B7-1protein and 5-fold dilutions of PD-L1 antibodies starting from 100 nM at50 μl were added to each well and incubated for 1 hour at 37° C. Theplates were washed with PBS/Tween and then incubate withStreptavidin-HRP for 1 hour at 37° C.

After washing, the plates were developed with TMB substrate and analyzedby spectrophotometer at OD 450 nm. As shown in FIGS. 10 and 11,Hu1210-41 can efficiently inhibit the binding of human PD-L1 to humanPD1 and B7-1.

1.9. Activity of Humanized Anti-PD-L1 Antibody to Promote Human T CellImmune Response

Mixed Lymphocyte Reaction Assay

To evaluate the in vitro function of humanized antibodies, the responseof human T cells assessed in a mixed lymphocyte reaction setting. HumanDCs were differentiated from CD14+monocytes in the presence of GM-CSFand IL-4 for 7 days. CD4+ T cells isolated from another donor were thenco-cultured with the DCs and serial dilutions of anti-PD-L1 blockingantibody. At day 5 post-inoculation, the culture supernatant was assayedfor IL-2 and IFNγ production. The results indicated that the Hu1210-8,Hu1210-9, Hu1210-16 and Hu1210-17 antibodies can dose-dependentlypromote IL-2 and IFNγ production, suggesting anti-PD-L1 antibodies canpromote human T cell response.

CMV Recall Assay

To evaluate the in vitro function of humanized antibodies, the responseof human T cells assessed in CMV recall assay. Human PBMCs werestimulated with 1 μg/ml CMV antigen in the presence of serious dilutedhumanized antibodies. As shown in FIGS. 12 and 13 the Hu1210-40,Hu1210-41 and Hu1210-17 can dose dependently promote the IFNγproduction.

1.10. Tumor Growth Inhibition by Anti-PD-L1 mAb.

Cells from the human lung adenocarcinoma cell line HCC827 will begrafted into NOD scid gamma (NSG) mice. NSG mice are NOD scid gammadeficient and the most immunodeficient mice making them ideal recipientsfor human tumor cell and PBMC grafting. 10 days post-graft, human PBMCswill be transplanted into the tumor-bearing mice. Approximately 20 dayspost-graft, once the tumor volume has reached 100-150 mm³, PD-L1antibody will be administered to the mice every other day at 5 mg/kg.Tumor volume will be monitored every other day in conjunction withantibody administration. As shown in FIG. 14, Hu1210-31 can inhibit thetumor growth by 30% at 5 mg/kg. Hu1210-41 antibody can dose-dependentlyinhibit the tumor growth, while the tumor weight was alsodose-dependently suppressed by Hu1210-41 antibody (FIG. 15).

1.11. Computer Simulation of Further Variation and Optimization of theHumanized Antibodies

It was contemplated that certain amino acid residues within the CDRregions or the framework regions could be changed to further improve orretain the activity and/or stability of the antibodies. Variants weretested, with a computational tool (VectorNTI, available atwww.ebi.ac.uk/tools/msa/clustalo/), with respect to their structural,conformational and functional properties, and those (within the CDRregions) that showed promises are listed in the tables blow.

TABLE 14 VH and VL CDRs and their variants suitable for inclusion inhumanized antibodies Name Sequence SEQ ID NO: VH CDR1 SYDMS 1 TYDMS 61CYDMS 62 SFDMS 63 SHDMS 64 SWDMS 65 SYDMT 66 SYDMC 67 VH CDR2TISDGGGYIYYSDSVKG 2 TISDGGAYIYYSDSVKG 68 TISDGGPYIYYSDSVKG 69TISDGGGFIYYSDSVKG 70 TISDGGGHIYYSDSVKG 71 TISDGGGWIYYSDSVKG 72TISDGGGYIYYSDTVKG 73 TISDGGGYIYYSDCVKG 74 TISDGGGYIYYSDSLKG 75TISDGGGYIYYSDSIKG 76 TISDGGGYIYYSDSMKG 77 VH CDR3 EFGKRYALDY 3QFGKRYALDY 78 DFGKRYALDY 79 NFGKRYALDY 80 EYGKRYALDY 81 EHGKRYALDY 82EWGKRYALDY 83 EFAKRYALDY 84 EFPKRYALDY 85 EFGRRYALDY 86 EFGKKYALDY 87EFGKRFALDY 88 EFGKRHALDY 89 EFGKRWALDY 90 VL CDR1 KASQDVTPAVA 4KATQDVTPAVA 91 KACQDVTPAVA 92 VL CDR2 STSSRYT 5 TTSSRYT 93 CTSSRYT 94SSSSRYT 95 SMSSRYT 96 SVSSRYT 97 STTSRYT 98 STCSRYT 99 STSTRYT 100STSCRYT 101 STSSKYT 102 STSSRFT 103 STSSRHT 104 STSSRWT 105 VL CDR3QQHYTTPLT 6 EQHYTTPLT 106 DQHYTTPLT 107 NQHYTTPLT 108 QEHYTTPLT 109QDHYTTPLT 110 QNHYTTPLT 111

(in Table 14, hotspot mutation residues and their substitutes areunderlined)

1.12. Identification of PD-L1 Epitope

This study was conducted to identify amino acid residues involved in thebinding of PD-L1 to the antibodies of the present disclosure.

An alanine-scan library of PD-L1 was constructed. Briefly, 217 mutantclones of PD-L1 were generated on Integral Molecular's proteinengineering platform. Binding of Hu1210-41 Fab to each variant in thePD-L1 mutation library was determined, in duplicate, by high-throughputflow cytometry. Each raw data point had background fluorescencesubtracted and was normalized to reactivity with PD-L1 wild-type (WT).For each PD-L1 variant, the mean binding value was plotted as a functionof expression (control anti-PD-L1 mAb reactivity). To identifypreliminary critical clones (circles with crosses), thresholds (dashedlines) of >70% WT binding to control MAb and <30% WT reactivity toHu1210-41 Fab were applied (FIG. 16). Y134, K162, and N183 of PDL1 wereidentified as required residues for Hu1210-41 binding. The lowreactivity of N183A clone with Hu1210-41 Fab suggests that it is themajor energetic contributor to Hu1210-41 binding, with lessercontributions by Y134 and K162.

The critical residues (spheres) were identified on a 3D PD-L1 structure,as illustrated in FIG. 17. These residues, Y134, K162, and N183,therefore, constitute an epitope of PD-L1 responsible for binding toantibodies of various embodiments of the present disclosure.

It is interesting to note that Y134, K162, and N183 are all locatedwithin the IgC domain of the PD-L1 protein. Both PD-1 and PD-L1'sextracellular portions have an IgV domain and an IgC domain. It iscommonly known that PD-L1 binds to PD-1 through bindings between theirIgV domains. Unlike such conventional antibodies, however, Hu1210-41binds to the IgC domain, which would have been expected to beineffective in inhibiting PD-1/PD-L1 binding. This different epitope ofHu1210-41, surprisingly, likely contributes to the excellent activitiesof Hu1210-41.

1.13. Antibody Engineering of Anti-PDL1 Antibody

Examples 1.13-1.15 attempted to identify further improved antibodiesbased on Hu1210-41 using mutagenesis.

Four sub-libraries were constructed for antibody engineering ofanti-PD-L1 monoclonal antibody, using either of the followingstrategies. In strategy 1, mutagenesis of heavy chain variable domain VHCDR3 or VL-CDR3 was perform by highly random mutation. In strategy 2,two CDR combination libraries composed of (VH-CDR3, VL-CDR3 and VL-CDR1)or (VH-CDR1, VH-CDR2 and VL-CDR2) were generated by CDR walking withcontrolled mutation rates.

Bio-Panning: the phage panning methods were adapted by shortening theincubation/binding time prior to the harsh washing condition. Briefly,100 μl magnetic streptavidin beads (Invitrogen, USA) were blocked with 1ml of MPBS for 1 hr at room temperature. In another tube, library phagewas pre-incubated (5×10{circumflex over ( )}11˜12 for each round) with100 μl magnetic streptavidin beads in 1 ml of MPBS to remove unwantedbinders. Magnet particle concentrator was used to separate the phage andbeads. The biotinylated PD-L1 protein was added to the phage andincubated 2 h at room temperature, and gently mixed using an over-headshaker. Beads carrying phage from the solution were separated in themagnetic particle concentrator and the supernatant was discarded. Thebeads were washed with fresh wash buffer, ten times with PBST and tentimes with PBS (pH7.4). 0.8 ml, 0.25% Trypsin in PBS (Sigma, USA) wasadded and incubated for 20 min at 37° C. to elute the phage. The outputphage was titrated and rescued for next round panning, decreasingantigen concentration round by round.

ELISA Screening and on/Off Rate Ranking

Clones were picked and induced from the desired panning output; phageELISA was conducted for primary screening; positive clones were analyzedby sequencing; unique hotspots were found. Table 15 shows the mutationsidentified. As shown below, the FGK residues in the CDRH3 are hotpotresidues producing improved antibodies.

TABLE 15 Mutations in the CDRs CDR-H1 (SEQ No.) CDR-H2 (SEQ No.) CDR-H3(SEQ No.) WT* SYDMS (1) TISDAGGYIYYRDSVKG (526) E FGK RYALD Y (3) B3SYDMS (1) TISDAGGYIYYRDSVKG (526) EFGKRYALDY (3) C4 SYDMS (1)TISDAGGYIYYRDSVKG (526) EFGKRYALD S (513) B1 SYDMS (1) TISDAGGYIYYRDSVKG(526) E IFN RYALDY (514) B6 SYDMS (1) TISDAGGYIYYRDSVKG (526) E LPWRYALDY (515) C3 SYDMS (1) TISDAGGYIYYRDSVKG (526) E LHF RYALDY (516) C6SYDMS (1) TISDAGGYIYYRDSVKG (526) E LYF RYALDY (517) A1 SYDMS (1)TISDAGGYIYYRDSVKG (526) E LLH RYALDY (518) A2 SYDMS (1)TISDAGGYIYYRDSVKG (526) E LRG RYALDY (519) A3 SYDMS (1)TISDAGGYIYYRDSVKG (526) EFGKRYALDY (3) CDR-L1 (SEQ No.) CDR-L2 (SEQ No.)CDR-L3 (SEQ No.) WT* KA S QDV T PAVA (4) STSSRYT (5) Q QH YTT PLT (6) B3KA K QDVTPAVA (520) STSSRYT (5) M QHYTTPLT (522) C4 KASQDV W PAVA (521)STSSRYT (5) QQH S TTPLT (523) B1 KASQDVTPAVA (4) STSSRYT (5) QQHYTTPLT(6) B6 KASQDVTPAVA (4) STSSRYT (5) OOHYTTPLT (6) C3 KASQDVTPAVA (4)STSSRYT (5) QQHYTTPLT (6) C6 KASQDVTPAVA (4) STSSRYT (5) OOHYTTPLT (6)A1 KASQDVTPAVA (4) STSSRYT (5) QQHYTTPLT (6) A2 KASQDVTPAVA (4) STSSRYT(5) QQHYTTPLT (6) A3 KASQDVTPAVA (4) STSSRYT (5) QQH SDA PLT (524) (*WTdiffers from Hu1210-41 by a S60R (Kabat numbering) substitution in theheavy chain to improve affinity.)

The amino acid sequences of the variable regions of these antibodies areshown in Table 16 below.

TABLE 16 Antibody sequences Name Sequence SEQ ID NO: WT-VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 493ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF GKRYALDYWGQGTTVTVSSWR-Vk DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS 494TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GRKLEIK B3-VHEVQLVESGGGLVQPGGSLRLSCAASGFT6FSSYDMSWVRQAPGKSLEWVAT 495ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF GKRYALDYWGQGTTVTVSSB3-Vk DIQMTQSPSSLSASVGDRVTITCKAKQDVTPAVAWYQQKPGKAPKLLIYS 496TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCMQHYTTPLTFGQ GTKLEIK C4-VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 497ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF GKRYALDSWGQGTTVTVSSC4-Vk DIQMTQSPSSLSASVGDRVTITCKASQDVWPAVAWYQQKPGKAPKLLIYS 498TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPLTFGQ GTKLEIK B1-VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 499ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTACYICAREI FNRYALDYWGQGTTVTVSSB1-Vk DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS 500TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK B6-VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 501ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREL PWRYALDYWGQGTTVTVSSB6-Vk DIQMTQSPSSLSASVGDRVTITCKASQDVTPACAWYQQKPGKAPKLLIYS 502TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK C3-VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 503ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTACYICAREL HFRYALDYWGQGTTVTVSSC3-Vk DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS 504TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK C6-VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 505ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREL YFRYALDYWGQGTTVTVSSC6-Vk DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS 506TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK A1-VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 507ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREL LHRYALDYWGQGTTVTVSSA1-Vk DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS 508TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK A2-VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 509ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREL RGRYALDYWGQGTTVTVSSA2-Vk DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS 510TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK A3-VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 511ISDAGGYIYYRDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF GKRYALDYWGQGTTVTVSSA3-Vk DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS 512TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSDAPLTFGQ GTKLEIK

1.14. Antigen Binding Properties of the PD-L1 Antibodies

As shown in Tables 15 and 16, totally 9 unique clones were characterizedand converted into full-length IgG.

Binding Property to Recombinant Human PD-L1

To evaluate the antigen binding activity, the antibodies were subjectedto ELISA test. Briefly, microtiter plates were coated with humanPD-L1-Fc protein at 2 μg/ml in PBS, 100 μl/well at 4° C. overnight, thenblocked with 100 μl/well of 5% BSA. 4-fold dilutions of humanizedantibodies starting from 10 μg/ml were added to each well and incubatedfor 1-2 hours at RT. The plates were washed with PBS/Tween and thenincubate with goat-anti-mouse IgG antibody conjugated with Horse RadishPeroxidase (HRP) for 1 hour at RT. After washing, the plates weredeveloped with TMB substrate and analyzed by spectrophotometer at OD450-630 nm. As shown in FIG. 18, all the humanized antibodies showedexcellent binding efficacy to human PD-L1, and B6 and C3 behaved betterthan the parental clone WT.

Binding Property to Mammalian Expressed Human PD-L1

To evaluate the antigen binding property, the antibodies were analyzedfor its binding to mammalian expressed PD-L1 by FACS. Briefly, PDL1-Rajicells were firstly incubated with 5-fold serious diluted humanizedantibodies starting at 2 □g/ml at RT for 1 hour. After wash by FACSbuffer (PBS with 2% FBS), the Alexa 488-anti-human IgG antibody wasadded to each well and incubated at RT for 1 hour. The MFI of Alexa 488was evaluated by FACSAriaIII. As shown in the FIG. 19, B6 highlyefficiently bound to PD-L1 expressed on mammalian cells, which was morepotent than the parental antibody WT.

Affinity ranking of humanized antibodies by Biacore

To explore the binding kinetics of the humanized antibody, this exampleperformed the affinity ranking using Biacore. As shown Table 17, B6, C3,C6, A1 and A3 showed better affinity than the parent antibody WT.

TABLE 17 Affinity ranking Antibody ka (1/Ms) kd (1/s) KD (M) WT 1.77E+054.64E−04 2.63E−09 B3 1.19E+05 2.96E−04 2.49E−09 C4 1.13E+05 5.06E−044.50E−09 B1 1.63E+05 2.61E−04 1.60E−09 B6 2.42E+05 2.46E−04 1.02E−09 C32.18E+05 2.99E−04 1.37E−09 C6 2.06E+05 3.34E−04 1.63E−09 A1 2.03E+052.76E−04 1 36E−09 A2 1.87E+05 4.75E−04 2.55E−09 A3 2.18E+05 3.24E−041.49E−09

1.15. Anti-PDL1 Antibody Cell-Based Function

To test the ability of anti-PDL1 antibodies to stimulate T cellresponse, hPD-1-expressed Jurkat cells were used. Briefly, Jurkat ishuman T cell leukemia cell line that can produce IL2 upon TCRstimulation. In this assay, Jurkat cells transfected with human PD-1gene by lentivirus were used as the responder cells. The Raji-PDL1 cellswere used as the antigen presenting cells (APC). StaphylococcalEnterotoxins (SE) are used to stimulate TCR signal. In this system,ectopically expressed huPDL1 can suppress SE stimulated IL-2 productionby Jurkat cells, while anti-PDL1 antibodies can reverse IL-2 production.In short, APCs (2.5×10⁴) were co-cultured with PD-1 expressing Jurkat Tcells (1×10⁵) in the presence of SE stimulation. Anti-PDL1 antibodies(starting from 100 nM and 1:4 serially diluted for 8 dose) were added atthe beginning of the culture. 48 hr later, culture supernatant wasevaluated for IL2 production by ELISA. As shown in FIG. 20, the B6monoclonal antibody was more potent than parental antibody WT.

Example 2. Preparation of Anti-LAG3 Monoclonal Antibodies

2.1. Screening of Full Human Monoclonal Antibodies Against LAG-3

Anti-LAG3 human monoclonal antibodies (α-LAG-3 mAbs) were generated byscreening full human Fab phage-display libraries. WildtypeLAG-3-ECD-huFc fragments can bind to Daudi cells while D1-D2 truncatedLAG-3-ECD-huFc fragments fail to bind Daudi cells (FIG. 21).Consequently, the D1-D2 domains are critical for LAG-3 function.

Antigens for phage-display library-panning. LAG-3 is a single-pass typeI membrane protein which belongs to the immunoglobulin (Ig) superfamilyand contains 4 extracellular Ig-like domains (ECD): domain (D)1, D2, D3and D4. A recombinant human LAG-3-ECD-human IgG1 (LAG-3-huFc) fusionprotein or a human D1-D2 truncated LAG-3-ECD-human IgG1(ΔD1D2-LAG-3-huFc) fusion protein were expressed in a 293T cell system.

Phage library. Ig gene segments in mammals are arranged in groups ofvariable (V), diversity (D), joining (J), and constant (C) exons. Thehuman Fab phage libraries were construed using the phage vectors, whichconsists of: 1) all human variable kappa (VK) repertoires; and 2) the VHof VH3-23 and VH1-69 germline genes, respectively, with geneticallyrandomized CDR3 regions from healthy human subjects.

Antigen screening and generation. To select the D1-D2 domain-specificphage binders, the phage libraries were subjected to antigen-basedpanning.

I) Phage Library Solution Panning Against LAG-3.

293F cells were transfected with a plasmid containing a D1-D2 deletedLAG-3 (ΔD1D2-LAG-3) sequence with a FLAG-tag at the N-terminus. At 3days post-transfection, the ΔD1D2-LAG-3 293F cells were used for phagelibrary screening. The phage libraries were performed the sequentialnegative screenings: streptavidin beads, ΔD1D2-LAG-3 transfected 293Fcells and biotin-labeled-human IgG1Fc protein. The resulting library wasthen incubated with biotinylated LAG-3-huFc LAG-3 for 2 hrs undermotion, followed by incubation with 100 μL of casein blockedstreptavidin-magnetic beads for 15 min. Unbound phages were removed bywashing with PBS 5-times. The bound phages were then eluted with freshlyprepared 100 mM triethylamine (TEA) and neutralized with the addition ofTris-HCl buffer. The resulting phages were labeled as the Output-1 phagelibraries. Output-1 phage libraries were subjected to the same screeningas described above to generate the Output-2 and subsequent Output-3phage libraries. Three rounds of phage library screening were performedin total.

II) Phage Library Immunotube Panning Against LAG-3

The phage libraries were used to perform sequential negative screenings:casein-coated immunotubes, ΔD1D2-LAG-3 transfected 293F cells and humanIgG1Fc protein. The resulting library was then incubated inLAG3-huFc-coated immunotubes for 2 hrs under motion. Unbound phages wereremoved by washing with PBST 5-20 times. Similar with cell-basedpanning, three rounds of phage library screening were performed intotal.

Output-3 phage libraries were diluted and plated to grow at 37° C. for 8hrs and captured by anti-kappa antibody-coated filters overnight at 22°C. Biotinylated LAG-3-huFc (50 nM) and NeutrAvidin-AP conjugate wereapplied to the filter to detect antigen binding anti-LAG3 phages.Positive phage plaques were picked and eluted into 100 μL of phageelution buffer. About 10-15 μL of eluted phages were then used to infect1 mL of XL1-Blue competent cells to make a high-titer (HT) phage forphage single point ELISA (SPE) (ELISA immobilized substrate coated with50 nM of each protein tested). 1×10¹⁰ plaque forming units (pfus) ofeach phage hit was used for SPE confirmation. The positive clones pickedfrom the filter lift were then tested for LAG-3 antigen binding withLAG-3-huFc and ΔD1D2-LAG-3-huFc. The D1-D2 specific binders wereamplified from antigen positive phages by PCR and sequenced. Ig lightchain V genes (VL) and VH sequences were analyzed to identify uniquesequences and determine sequence diversity.

VL and VH gene sequences of all hits were cloned into expression vectorspFUSE2ss-CLIg-hk (light chain, InvivoGen Cat No. pfuse2ss-hclk) andpFUSEss-CHlg-hG1 (heavy chain, InvivoGen Cat No. pfusess-hchg1). Theantibodies were expressed in HEK293 cells and purified using Protein APLUS-Agarose. Sequences of the antibodies and their CDR regions areprovided in the table below.

TABLE 18 heavy chain variable regions Antibody No. VH SEQ ID NO:NLAG3-HDB169-T03 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 254IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGS SWFDYWGQGTLVTVSSNLAG3-HDB169-T05 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 255IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCASSY HGGGYHRYWGQGTLVTVSSNLAG3-HDB169-T06 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 256IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTTSK YSGSALRYWGQGTLVTVSSNLAG3-HDB169-T07 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 257IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDR TGAFDYWGQGTLVTVSSNLAG3-HDB169-T08 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 258IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARHE TVAGSFDYWGQGTLVTVSSNLAG3-HDB169-T10 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 259IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARTGYYGGNSGAFDIWGQGTMVTVSS NLAG3-HDB169-T13QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 260IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARAG TGMDLVFNSWGQGTLVTVSSNLAG3-HDB169-T23 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 261IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGL ARGDLNFGYWGQGTLVTVSSNLAG3-HDB169-S24 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 262IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTREP HFDYWGQGTLVTVSSNLAG3-HDB169-S27 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 263IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTTAA PGSYYLVFHYWGQGTLVTVSSNLAG3-HDB169-S31 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 264IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDA GPVGYYGMDVWGQGTTVTVSSNLAG3-HDB169-S32 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 265IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAGDG LYGSGSFGYWGQGTPVTVSSNLAG3-HDB169-S61 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 266IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAKDI RWFYGMDVWGQGTTVTVSSwNLAG3-HDB169-S64 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 267IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARHE SGIAGGHFDYWGQGTLVTVSSNLAG3-HDB169-S86 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 268IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSELTAVYYCARDA GPVGYYGMDVWGQGTTVTVSSNLAG3-HDB169-S87 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 269IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAKDI RWYYGMDVWGQGTTVTVSSNLAG3-HDB169-T94 QVQLVQSGAEVKKPGSSVKVFCKASGGTFSSYAISWVRQAPGQGLEWMGG 270IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAKGVRGTYQIGYYGMDVWGQGTTVTVSS NLAG3-HDB169-T97QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 271IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARQG TAMALDYWGQGTLVTVSSNLAG3-HDB169-T99 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 272IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCVRDL QDWNYGGAAYWGQGTLVTVSSNLAG3-HDB169-S103 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 273IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDD YYYGQFDSWGQGTLVTVSSNLAG3-HDB169-S107 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 274IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREI TGTSYTALDSWGQGTLVTVSSNLAG3-HDB169-S109 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 275IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGH IDGQAAGDYWGQGTLVTVSSNLAG3-HDB169-S119 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 276IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAAST LRVPNPPYWGQGTLVTVSSNLAG3-HDB169-S120 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 277IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSG DRYDFWSGYWGQGTLVTVSSNLAG3-HDB169-S127 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 278IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAAST LRVPNPPYWGQGTLVTVSSNLAG3-HDB169-S128 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 279IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDA GPCGYYGMDVWGQGTMVTVSSNLAG3-HDB169-S136 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 280IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTRGQ DSTWYSSFDYWGQGTLVTVSSNLAG3-HDB169-S139 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 281IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAAST LRLPNPPYWGQGTLVTVSSNLAG3-HDB169-S150 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 282IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAAST TSFYSHGMDVWGQGTTVTVSSNLAG3-HDB169-S157 QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS 283GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVRKT PFWGALDSWGRGTLVTVSSNLAG3-HDB169-S164 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG 284IIPIFGTANYAQKFQGRVTFTADESTSTAYMELSSLRSEDTAVYYCARGF TYGDFFFDYWGQGTLVTVSSNLAG3-HDB169-S177 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAPSWVRQAPGQGLEWMGG 285IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDV RGVTYLGMDVWGQGTTVTVSSNLAG3-HDB323-S20 QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS 286GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVRKT PFWGTLDSWGRGTLVTVSSNLAG3-HDB323-S21 QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS 287GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVRRT PFWGALDSWGRGTLVTVSSNLAG3-HDB323-S32 QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS 288GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVRKT PFWGALDSWGRGTLVTVSSNLAG3-HDB323-S35 QLLESGGGLVQPGGSLRLSCLAASGFTFSSYAMSWVRQAPGKGLEWSAIS 289GSGGSTYYADSYTLGRFTLSRDNSKNTLYLQMNSLRAEDTAVYYCAKRKGLGSPTDYYYGMDVWGQGTTVTVSS NLAG3-HDB323-S52QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS 290GSGGSTYYADSVKGRFTTSRDNSKNTLYLQMNSLRAEDTAVYYCARVRKT PFWGALDSWGRGTLVTVSSNLAG3-HDB323-S55 QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS 291GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVRKT PFWGTLDSWGRGSLVTVSSNLAG3-HDB323-T89 QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS 292GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPEYD YYYGMDVWGQGTTVTVSSNLAG3-HDB323-T92 QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS 293GSGGSTYYADSVKGRFTISRDN3KNTLYLQMNSLRAEDTAVYYCAKGGGS YDYWGQGTLVTVSSNLAG3-HDB323-T94 QLLESGGGLVQVGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS 294GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARALNG MDVWGQGTMVTVSSNLAG3-HDB323-S102 QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS 295GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRPLQGTAAADSYYYYAMDVWGQGTTVTVSS NLAG3-HDB323-S103QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS 296GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLHSY LSEEFDPWGQGTLVTVSSNLAG3-HDB323-S107 QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS 297GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVRKT PFWGALDSWGRGTLVTVSSNLAG3-HDB323-S114 QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS 298GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLSAV NTYIDDWGQGTLVTVSSNLAG3-HDB323-S135 QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS 299GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVTKT PFWGTLDYWGQGTLVTVSSNLAG3-HDB323-S143 QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS 300GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVRRT PFWGALDSWGRGTLVTVSSNLAG3-HDB323-S146 QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS 301GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSQS PVWGYFDYWGQGMLVTVSSNLAG3-HDB323-S161 QLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS 302GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGYY DFWSGYSDYWGQGTLVTVSS

TABLE 19 Heavy Chain CDRs Antibody No. CDR H1 SEQ ID NO: CDR H2 SEQ IDNO: CDR H3 SEO ID NO: NLAG3-HDB169-T03 SYAIS 116 GIIPIFGTANYAQKFQG 118ARGSSWFDY 120 NLAG3-HDB169-T05 SYAIS 116 GIIPIFGTANYAQKFQG 118ASSYHGGGYHRY 121 NLAG3-HDB169-T06 SYAIS 116 GIIPIFGTANYAQKFQG 118TTSKYSGSALRY 122 NLAG3-HDB169-T07 SYAIS 116 GIIPIFGTANYAQKFQG 118ARDRTGAFDY 123 NLAG3-HDB169-T08 SYAIS 116 GIIPIFGTANYAQKFQG 118ARHETVAGSFDY 124 NLAG3-HDB169-T10 SYAIS 116 GIIPIFGTANYAQKFQG 118ARTGYYGGNSGAFDI 125 NLAG3-HDB169-T13 SYAIS 116 GIIPIFGTANYAQKFQG 118ARAGTGMDLVFNS 126 NLAG3-HDB169-T23 SYAIS 116 GIIPIFGTANYAQKFQG 118ARGLARGDLNFGY 127 NLAG3-HDB169-S24 SYAIS 116 GIIPIFGTANYAQKFQG 118TREPHFDY 128 NLAG3-HDB169-S27 SYAIS 116 GIIPIFGTANYAQKFQG 118TTAAPGSYYLVFHY 129 NLAG3-HDB169-S31 SYAIS 116 GIIPIFGTANYAQKFQG 118ARDAGPVGYYGMDV 130 NLAG3-HDB169-S32 SYAIS 116 GIIPIFGTANYAQKFQG 118AGDGLYGSGSFGY 131 NLAG3-HDB169-S61 SYAIS 116 GIIPIFGTANYAQKFQG 118AKDIRWFYGMDV 132 NLAG3-HDB169-S64 SYAIS 116 GIIPIFGTANYAQKFQG 118ARHESGIAGGHFDY 133 NLAG3-HDB169-S86 SYAIS 116 GIIPIFGTANYAQKFQG 118ARDAGPVGYYGMDV 130 NLAG3-HDB169-S87 SYAIS 116 GIIPIFGTANYAQKFQG 118AKDIRWYYGMDV 134 NLAG3-HDB169-T94 SYAIS 116 GIIPIFGTANYAQKFQG 118AKGVRGTYQIGYYGMDV 135 NLAG3-HDB169-T97 SYAIS 116 GIIPIFGTANYAQKFQG 118ARQGTAMALDY 136 NLAG3-HDB169-T99 SYAIS 116 GIIPIFGTANYAQKFQG 118VRDLQDWNYGGAAY 137 NLAG3-HDB169-S103 SYAIS 116 GIIPIFGTANYAQKFQG 118ARDDYYYGQFDS 138 NLAG3-HDB169-S107 SYAIS 116 GIIPIFGTANYAQKFQG 118AREITGTSYTALDS 139 NLAG3-HDB169-S109 SYAIS 116 GIIPIFGTANYAQKFQG 118ARGHIDGQAAGDY 140 NLAG3-HDB169-S119 SYAIS 116 GIIPIFGTANYAQKFQG 118AASTLRVPNPPY 141 NLAG3-HDB169-S120 SYAIS 116 GIIPIFGTANYAQKFQG 118ARSGDRYDFWSGY 142 NLAG3-HDB169-S127 SYAIS 116 GIIPIFGTANYAQKFQG 118AASTLRVPNPPY 141 NLAG3-HDB169-S128 SYAIS 116 GIIPIFGTANYAQKFQG 118ARDAGPVGYYGMDV 130 NLAG3-HDB169-S136 SYAIS 116 GIIPIFGTANYAQKFQG 118TRGQDSTWYSSFDY 143 NLAG3-HDB169-S139 SYAIS 116 GIIPIFGTANYAQKFQG 118AASTLRLPNPPY 144 NLAG3-HDB169-S150 SYAIS 116 GIIPIFGTANYAQKFQG 118ATTQTSFYSHGMDV 145 NLAG3-HDB169-S157 SYAIS 116 GIIPIFGTANYAQKFQG 118ARVRKTPFWGALDS 146 NLAG3-HDB169-S164 SYAIS 116 GIIPIFGTANYAQKFQG 118ARGFTYGDFIFDY 147 NLAG3-HDB169-S177 SYAIS 116 GIIPIFGTANYAQKFQG 118ARDVRGVTYLGMDV 148 NLAG3-HDB323-S20 SYAMS 117 AISGSGGSTYYADSVKG 119ARVRKTPFWGTLDS 149 NLAG3-HDB323-S21 SYAMS 117 AISGSGGSTYYADSVKG 119ARVRRTPFWGALDS 150 NLAG3-HDB323-S32 SYAMS 117 AISGSGGSTYYADSVKG 119ARVRKTPFWGALDS 146 NLAG3-HDB323-S35 SYAMS 117 AISGSGGSTYYADSVKG 119AKRKGLGSPTDYYYGMDV 151 NLAG3-HDB323-S52 SYAMS 117 AISGSGGSTYYADSVKG 119ARVRKTPFWGALDS 146 NLAG3-HDB323-S55 SYAMS 117 AISGSGGSTYYADSVKG 119ARVRKTPFWGTLDS 149 NLAG3-HDB323-T89 SYAMS 117 AISGSGGSTYYADSVKG 119VRPEYDTYYYGMDV 152 NLAG3-HDB323-T92 SYAMS 117 AISGSGGSTYYADSVKG 119AKGGGSYDY 153 NLAG3-HDB323-T94 SYAMS 117 AISGSGGSTYYADSVKG 119 ARALNGMDV154 NLAG3-HDB323-S102 SYAMS 117 AISGSGGSTYYADSVKG 119TRPLQGIAAADSYYYYAMDV 155 NLAG3-HDB323-S103 SYAMS 117 AISGSGGSTYYADSVKG119 ARLHSYLSEEFDP 156 NLAG3-HDB323-S107 SYAMS 117 AISGSGGSTYYADSVKG 119ARVRKTPFWGALDS 146 NLAG3-HDB323-S114 SYAMS 117 AISGSGGSTYYADSVKG 119AKLSAVNTYIDD 157 NLAG3-HDB323-S135 SYAMS 117 AISGSGGSTYYADSVKG 119ARVTKTPFWGTLDY 158 NLAG3-HDB323-S143 SYAMS 117 AISGSGGSTYYADSVKG 119ARVRRTPFWGALDS 150 NLAG3-HDB323-3146 SYAMS 117 AISGSGGSTYYADSVKG 119ARVSQSPVWGYFDY 159 NLAG3-HDB323-S161 SYAMS 117 AISGSGGSTYYADSVKG 119AKDGYYDFWSGYSDY 160

TABLE 20 Light chain variable regions Antibody No. VL SEQ ID NO:NLAG3-HDB169-T03 DIQLTQSPSSLSAFVGDRVTITCQANQDIHHYLNWYQQKPGKAPKLLIYD 303ASILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQADSFPITFGQ GTRLEIKRNLAG3-HDB169-T05 EIVLTQSPDSLAVSLGERATINCKSSQSVLYSSSNKNYLAWYQQKPGQPP 304KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYST PWTFGPGTKLEIKRNLAG3-HDB169-T06 DIQMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGHPP 305KLLVYWASTRESGVPARFSASGSGTDFTLAISNLQAEDVAVYYCQQYYST PWTFGQGTKVEIKRNLAG3-HDB169-T07 EIVLTQSPLSLPVTPGEPASISCRSSQNLLHSDGYNYLNWYLQKPGQSPQ 306LLIYLGSNRATGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP WTFGQGTKVEIKRNLAG3-HDB169-T08 DIVMTQSPDSLAVSLGERATINCKSSQSVLYTSNNKNYLAWYQQKPGQPP 307KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAIYYCQQYYST PWTFGQGTKLEIKRNLAG3-HDB169-T10 AIQLTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPP 308KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDSATYYCQQSFTT PWTFGQGTKVEIKRNLAG3-HDB169-T13 DIQMTQSPSSLSASVGDRVTITCQASQDINRYLSWYQQKPGKAPKLLIYD 309ASNLETGVPSRFSGSASGTDFTFAISSLQPEDIATYYCQQYDNLPPTFGQ GTRLEIKRNLAG3-HDB169-T23 EIVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYA 310ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFASYYCQQSYGSPVTFGQ GTKLEIKRNLAG3-HDB169-S24 EIVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYD 311ASNLETGVPSRFSGSGSGTEFTLTISSLRPEDFATYFCQQADSFPITFGQ GTRLEIKRNLAG3-HDB169-S27 DIQLTQSPSSLSASVGDRVTITCRASQTISSHLNWYQQKPGKAPKVLIYA 312ASSLQSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQGNSFPFTFGP GTKVEIKRNLAG3-HDB169-S31 AIRMTQSPSTLSASVGDRVTITCRASQGIAGWLAWYQQKPGKAPKLLIYA 313ASSLQSGVPSRFSGSASGTDFTLTISNLQPEDFATYYCQQAKSFPLTFGG GTKVEIKRNLAG3-HDB169-832 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPQPP 314KLLIYWASTRESGVPDRFSGTGSGTDFTLTISSLQAEDVAVYYCQQSYST PWTFGQGTKLEIKNLAG3-HDB169-861 DIVMTQSPSSVSAFVGDRVTITCRASQGVSSWLAWFQQKPGKAPKLLIYA 315ASTLQSGVPSRFSGRGYGTEFTLTISSLQPEDLATYYCQQVKSFPLTFGG GTKVDIKRNLAG3-HDB169-S64 DIVMTQSPDSLAVSLGERATINCKSSQSLFYHSNNHNYLAWYQQKPGQPP 316KLLIYWASTRQSGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQYYNT PWTFGQGTKVEIKRNLAG3-HDB169-S86 AIRMTQSPSTLSASVGDRVTITCRASQGIAGWLAWYQQKPGKAPKLLIYA 317ASSLQSGVPSRFSGSASGTDFTLTISNLQPEDFATYYCQQAKSFPLTFGG GTKVEIKRNLAG3-HDB169-S87 DIVMTQSPSSVSAFVGDRVTITCRASQGVSSWLAWFQQKPGKAPKLLIYA 318ASTLQSGVPSRFSGRGYGTEFTLTISSLQPEDLATYYCQQVKSFPLTFGG GTKVDIKRNLAG3-HDB169-T94 DIVMTQSPSSLSASVGDRVTITCRASQGISSSLAWYQQKPGKAPNLLIYT 319ASTLQNGVPSRFSGSGSGTDFTLTISGLQPEDFATYYCQQTKNFPLTFGQ GTRLEIKRNLAG3-HDB169-T97 EIVLTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQRPGQPP 320KLLISWASTRESGVPDRFSGSGSGADFSLTISSLQAEDVAVYYCQQYYST PWTFGQGTKLEIKRNLAG3-HDB169-T99 VIWMTQSPSSLSASVGDSVTITCQASRDISNSLSWHQQKPGKAPKLLIYA 321ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTKSFPLTFGG GTKVEIKRNLAG3-HDB169-S103 EIVMTQSPSSLSASVGDRVTISCRASQSISRYLNWYQQKPGQAPKLLIYA 322AFSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPRTFGQ GTKLEIKRNLAG3-HDB169-S107 DVVMTQSPSTVSASVGDRITITCRASRSISNWLAWYQQKPGKAPKLLIYA 323ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKSFPLTFGG GTKVEIKNLAG3-HDB169-S109 DIQLTQSPDSLAVSLGERATINCKSSQSVFYRSNQKNYLAWYQQKPGQTP 324RLLIYGASSRATGIPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRA PWTFGQGTKVEIKRNLAG3-HDB169-S119 EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYG 325ISSRATGIPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQANNFPLTFGG GTKLEIKRNLAG3-HDB169-S120 EIVLTQSPSSVSASVGDRVTITCRASRGISSWLAWYQQKPGKAPKLLIYA 326ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKSFPLTFGG GTKVEIKRNLAG3-HDB169-S127 EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYG 327ISSRATGIPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQANNFPLTFGG GTKLEIKRNLAG3-HDB169-S128 AIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYA 328ASSLQSGVPSRFSGSGSGTDFTLTISRLQPEDFATYYCQQAKSFPLTFGG GTKVEIKRNLAG3-HDB169-S136 AIRMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPNLLIYA 329VSTLQSGVPSRFSGSGSGTVFTLTISSLQPEDFATYFCQQGNSFPLTFGG GTKVEIKRNLAG3-HDB169-S139 DIQLTQSPSTLSASVGDRVTITCRASQAISNLLAWYQQKPGKPPNLLIYD 330ISTLQNGVPSRFSGSGSGTDFTLTINSLQPEDFAIYYCQQSKNFPVTFGG GTKVEIKRNLAG3-HDB169-S150 DIQLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYG 331ASTLQSGVPSRFSGSGSGADYTLTISSLQPEDFATYYCQQANSFPLTFAG GTKLEIKRNLAG3-HDB169-S157 DIQLTQSPSSLSASPGDRVTITCRASQGISTWLAWYQQKPGNAPKLLIYA 332ASSLQSGVPSRFSGSKSGTEYTLTISSLQPEDFATYYCQQLESYPLTFGG GTKVEIKRNLAG3-HDB169-S164 AIRMTQSPDSLVVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPP 333KLLIYWASTRESGVPDRFSGSGSGTDFTLSISSLQAEDVAVYYCQQYYSS PTFGGGTKVEIKRNLAG3-HDB169-S177 DVVMTQSPFFLSASVGDRVTITCRASQGIASNLAWYQQKPGKAPKLLIYA 334ASTLQSGVPSRFTGSGSGTEFTLTVTSLQPEDFATYYCQQLKTFPLTFGG GTKVEIKRNLAG3-HDB323-S20 VIWMTQSPSSLSASVGDRVTITCRASQGVSSYLAWYQQKPGKAPKLLIYA 335ASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQTNWFPLTFGP GTRLEIKRNLAG3-HDB323-S21 DIQMTQSPSSLSTSAGDTVTITCRASQSIYTYLNWYQQKPGKAPNLLIYG 336ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAQSFPITFGQ GTRLEIKRNLAG3-HDB323-S32 VIWMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYA 337ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHSFPLTFGG GTKVEIKRNLAG3-HDB323-S35 AIQLTQSPSTLSASVGDRVTITCRASQFVSDWLAWYQQKPGKAPKLLIYA 338ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCLQDYHFPLTFGG GTKLEIKRNLAG3-HDB323-S52 DVVMTQSPSSVSASVGDRVTITCRASQDIVNWLAWYQQKPGKAPKLLIYA 339ASTLESGAPSRFSASGSGTDFTLTISSLQPDDFATYYCQQGHSFPLTFGP GTKLEIKRNLAG3-HDB323-S55 DIVMTQSPSSLSASVGDRVTITCRASQSIYTYLNWYQQKPGKAPKLLIYD 340ASSLQSGVPSRFSGSGYGTEFTLTISGLQPEDFATYYCQOSYIFPLTFGR GTKVEIKRNLAG3-HDB323-T89 AIRMTQSPSFVSASVGDRVTIACRASQTISTWLAWYQQKPGKAPKVLISK 341ASNLQSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDTYWTFGQG TKVEIKRNLAG3-HDB323-T92 AIRMTQSPSFVSASVGDRVTIACRASQTISTWLAWYQQKPGKAPKVLISK 342ASNLQSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDTYWTFGQG TKVEIKRNLAG3-HDB323-T94 DIVMTQSPSFVSASVGDTVTITCRASQGISSYLAWYQQKPGKAPKLLIYA 343ASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPEFTFG PGTKVEIKRNLAG3-HDB323-S102 DIQMTQSPSTLSASVGDRVTITCRASQSIGYWLAWYQQKPGKAPKLLIYR 344ASSLQSGVPSRFSGSGSATEFTLTITSLQPDDFATYFCQQYSSYWTFGQG TKVEIKRNLAG3-HDB323-S103 EIVLTQSPSSLSASVGDTVTITCRATQSISSWLAWYQQKPGKAPQRLISG 345ASTLQSGVPSRFSGSGSGTEFTLTISGLQPEDFATYYCLQHNTYPFTFGQ GTKVEIKRNLAG3-HDB323-S107 DIVMTQSPSSVSASVGDRVTITCRASQGVRNWLAWYQQKPGKAPKLLIYA 346ASHLQSGVPSRFSGSGSGTDFTLTISSLQTDDFATYYCQQGHSFPLTFGG GTKVEIKRNLAG3-HDB323-S114 DIVMTQSPSSVSASVGDRVTITCRASQGVRNWLAWYQQKPGKAPKLLIYA 347ASHLQSGVPSRFSGSGSGTDFTLTISSLQTDDFATYYCQQGHSFPLTFGG GTKVEIKRNLAG3-HDB323-S135 VIWMTQSPSTLSASVGDRVTITCRASQSINNYLAWYQQKPGKAPKLLIYD 348ASTLQSGVPSRFSGGGSGTDFTLTINSLQPDDFASYYCQQAHSFPFTFGG GTKLEIKRNLAG3-HDB323-S143 EIVMTQSPSSVSASVGDRVTITCRASQDITSWLAWYQQKPGKAPKLLIYA 349ASTLESGVPSRFSGSGSGTDFTLTITGLQPEDFATYYCQQANMFPLTFGG GTKVEIKRNLAG3-HDB323-S146 AIRMTQSPSSLSASVGDRVTITCRASQGIYDYLAWYQQKPGKAPSLLIYA 350ASNLERGVPSRFSGSGSGKYFILTISSLQPEDFATYYCQQANSFPLTFGG GTKVEIKRNLAG3-HDB323-S161 AIQLTQSPSSLSASVGDRVTITCRASEGISGWLAWYQQIPGKAPKLLIYA 351ASSLETGVPSRFSGSGYGTDFTLTISSLQPEDFATYYCQQADSFPFTFGP GTKVEIKR

TABLE 21 Light Chain CDRs Antibody No. CDR L1 SEQ ID NO: CDR L2 SEQ IDNO: CDR L3 SEQ ID NO: NLAG3-HDB169-T03 QANQDIHHYLN 161 DASILQS 196QQADSFPIT 218 NLAG3-HDB169-T05 KSSQSVLYSSSNKNYLA 162 WASTRES 197QQSYSTPWT 219 NLAG3-HDB169-T06 KSSQSVLYSSNNKNYLA 163 WASTRES 197QQYYSTPWT 220 NLAG3-HDB169-T07 RSSONLLHSDGYNYLN 164 LGSNRAT 198QQSYSTPWT 219 NLAG3-HDB169-T08 KSSQSVLYTSNNKNYLA 165 WASTRES 197QQYYSTPWT 220 NLAG3-HDB169-T10 KSSQSVLYSSNNKNYLA 163 WASTRES 197QQSFTTPWT 221 NLAG3-HDB169-T13 QASQDINRYLS 166 DASNLET 199 QQYDNLPPT 222NLAG3-HDB169-T23 OASODISNYLN 167 AASSLQS 200 QQSYGSPVT 223NLAG3-HDB169-S24 QASODLSNYLN 167 DASNLET 199 QQADSFPIT 218NLAG3-HDB169-S27 RASQTISSHLN 168 AASSLQS 200 QQGNSFPFT 224NLAG3-HDB169-S31 RASQGIAGWLA 169 AASSLQS 200 QQAKSFPLT 225NLAG3-HDB169-S32 KSSQSVLYSSNNKNYLA 163 WASTRES 197 QQSYSTPWT 219NLAG3-HDB169-S61 RASQGVSSWLA 170 AASTLQS 201 QQVKSFPLT 226NLAG3-HDB169-S64 KSSQSLFYHSNNHNYLA 171 WASTRQS #N/A QQYYNTPWT 227NLAG3-HDB169-S86 RASQGIAGWLA 169 AASSLQS 200 QQAKSFPLT 225NLAG3-HDB169-S87 RASQGVSSWLA 170 AASTLQS 201 QQVKSFPLT 226NLAG3-HDB169-T94 RASQGISSSLA 172 TASTLQN 212 QQTKNFPLT 228NLAG3-HDB169-T97 KSSQSVLYSSNNKNYLA 163 WASTRES 197 QQYYSTPWT 220NLAG3-HDB169-T99 QASRDISNSLS 173 AASSLQS 200 QQTKSFPLT 230NLAG3-HDB169-S103 RASQSISRYLN 174 AAFSLQS 202 QQSYNTPRT 231NLAG3-HDB169-S107 RASRSISNWLA 175 AASSLQS 200 QQAKSFPLT 225NLAG3-HDB169-S109 KSSQSVFYRSNQKNYLA 176 GASSRAT 203 QQSYRAPWT 232NLAG3-HDB169-S119 RASQSVSSYLA 177 GISSRAT 204 QQANNFPLT 233NLAG3-HDB169-S120 RASRGISSWLA 178 AASTLQS 201 QQAKSFPLT 225NLAG3-HDB169-S127 RASQSVSSYLA 177 GISSRAT 204 QQANNFPLT 233NLAG3-HDB169-S128 RASQGISSWLA 179 AASSLQS 200 QQAKSFPLT 225NLAG3-HDB169-S136 RASQSISSYLN 180 AVSTLQS 205 QQGNSFPLT 234NLAG3-HDB169-S139 RASQAISNLLA 181 DISTLQN 206 QQSKNFPVT 235NLAG3-HDB169-S150 RASQGISSWLA 179 GASTLQS 207 QQANSFPLT 236NLAG3-HDB169-S157 RASQGISTWLA 182 AASSLQS 200 QQLESYPLT 237NLAG3-HDB169-S164 KSSQSVLYSSNNKNYLA 163 WASTRES 197 QQYYSSPT 238NLAG3-HDB169-S177 RASQGIASNLA 183 AASTLQS 201 QQLKTFPLT 239NLAG3-HDB323-S20 RASQGVSSYLA 184 AASSLQS 200 QQTNWFPLT 240NLAG3-HDB323-S21 RASQSIYTYLN 185 GASSLQS 208 QQAQSFPIT 241NLAG3-HDB323-S32 RASQGISSWLA 179 AASSLQS 200 QQAHSFPLT 242NLAG3-HDB323-S35 RASQFVSDWLA 186 AASTLQS 201 LQDYHFPLT 243NLAG3-HDB323-S52 RASQDIVNWLA 229 AASTLES 209 QQGNSFPLT 244NLAG3-HDB323-S55 RASQSIYTYLN 185 DASSLQS 210 QQSYIFPLT 245NLAG3-HDB323-T89 RASQTISTWLA 187 KASNLQS 211 QQYDTYWT 246NLAG3-HDB323-T92 RASQTISTWLA 187 KASNLQS 211 QQYDTYWT 246NLAG3-HDB323-T94 RASQGISSYLA 188 AASTLQS 201 QQLNSYPLFT 247NLAG3-HDB323-S102 RASQSIGYWLA 189 RASSLQS 213 QQYSSYWT 248NLAG3-HDB323-S103 RATQSISSWLA 190 GASTLQS 207 LQHNTYPFT 249NLAG3-HDB323-S107 RASQGVRNWLA 191 AASHLQS 214 QQGHSFPLT 244NLAG3-HDB323-S114 RASQGVRNWLA 191 AASHLQS 214 QQGHSFPLT 250NLAG3-HDB323-S135 RASQSINNYLA 192 DASTLQS 215 QQAHSFPFT 251NLAG3-HDB323-S143 RASQDITSWLA 193 AASTLES 209 QQANMFPLT 252NLAG3-HDB323-S146 RASQGIYDYLA 194 AASNLER 216 QQANSFPLT 236NLAG3-HDB323-S161 RASEGISGMLA 195 AASSLET 217 QQADSFPFT 253

2.2. The Binding of Human Anti-LAG3 Antibodies to LAG3 Protein Derivedfrom Various Species.

To evaluate the capability of the anti-LAG-3 antibodies to bind tohuman, rat, and mouse LAG3 the antibodies identified in Example 2.1 wereevaluated for their binding property through ELISA. The human, rat andmouse LAG3 ECD-Fc protein were coated to ELISA plate at 1 μg/ml with 100μl/well. Antibodies from Example 1 were serially diluted with ELISAdiluent buffer. To assess binding, LAG-3 antibodies at variousconcentrations 10 μg/ml, 3.333 μg/ml, 1.111 μg/ml, 0.370 μg/ml, 0.123μg/ml, 0.041 μg/ml, 0.014 μg/ml, 0.005 μg/ml, 0.0015 μg/ml and 0.0005μg/ml) were then added to LAG3 antigen coated plate for 1.5 hr RT. Theresulting plates were washed and then labeled with anti-humanIgG(Fab)-HRP antibody. The S31 can only bind to human LAG3. The S27 andT99 can bind to human LAG3 and rat/mouse LAG3 with lower potency. TheS119 antibody can bind to human, rat and mouse LAG3 at high potency(FIGS. 22A-22D).

2.3. The Binding of Human Anti-LAG3 Antibodies to Cell Surface LAG-3Antigen on Activated Human Primary CD4+ T Cells.

LAG-3 is expressed on activated or exhausted T cells. CD4+ T cells wereisolated using CD4 magnetic beads. The purified human CD4+ T cells werestimulated with Dynabeads® Human T-Activator CD3/CD28 for 72 hrs.Antibodies from Example 2.1 were serially diluted with FACS buffer. Toassess binding, LAG-3 antibodies at various concentrations (10 μg/ml,3.333 μg/ml, 1.111 μg/ml, 0.370 μg/ml, 0.123 μg/ml, 0.041 μg/ml, 0.014μg/ml and 0.005 μg/ml) were then added to the activated human CD4 Tcells in the presence of mouse anti-human LAG3 PE antibody (eBioscience,clone: 3DS223H) for 30 min on ice. The labeled cells were washed withFACS buffer and subsequently labeled with APC-conjugated anti-human IgGantibodies for 30 min on ice. The resulting cells were washed once withFACS buffer. Labeled cells were evaluated for fluorescence intensity byflow cytometry in a BD FACSCalibur™. As shown in FIG. 23, the S27, S31,T99 and S119 antibodies can dose-dependently bind to LAG3 expressed onthe activated human CD4+ T cells.

2.4. Anti-LAG-3 Antibody Inhibition of Soluble LAG-3 (sLAG) Binding toMHC Class II Receptor

To evaluate the ability of anti-LAG-3 antibodies to block the binding ofsLAG-3 to MHC class II receptor, an in vitro binding assay was designedusing biotin-labeled LAG-3-ECD-huFc fusion proteins and Raji cellsexpressing MHC class II receptor. Antibodies from Example 1 wereserially diluted from 20 μg/mL with FACS buffer and pre-incubated with 6μg/mL of biotin-LAG-3-ECD-huFcc for 30 min at room temperature. Theantibody mixture was then added to FcR blocked Raji cells and incubatedfor 30 min on ice. Cells were then washed with FACS buffer andsubsequently stained with streptavidin PE for 30 min on ice andsubsequently washed once with FACS buffer. Labeled cells were evaluatedfor fluorescence intensity by flow cytometry in a BD FACSCalibur™. Asshown in FIG. 24, the S27, S31, S119 and T99 antibodies can dosedependently inhibit the binding of LAG3 to its receptor MHC class IImolecules.

2.5. Stimulation of IL-2 Production in Peripheral Blood MononuclearCells (PBMCs) by Anti-LAG-3 Antibodies.

Staphylococcal enterotoxin B (SEB) is a superantigen that simultaneouslybinds to MHC class II antigens and T cell receptors (TCRs), bringingthem together in such a way as to induce T cell proliferation andcytokine production. 2×10⁵ PBMCs were stimulated with SEB in thepresence of the antibodies from Example 1 at various concentrationsstarting from 20 μg/ml at 1:3 serial dilutions for 6 doses. Three dayslater, IL-2 concentration in the culture supernatant was evaluated byELISA. As shown in FIG. 25, similar to PD-1 antibody, anti-LAG3antibodies (S24, S27, S31, S87, S119, T99 and S20) can dose dependentlyenhanced IL-2 production as compared with SEB stimulation only.

2.6. Reversing the Inhibition of Regulatory T Cells (T_(reg)) onEffector T Cells (T_(effs)) Using Anti-LAG-3 Antibodies.

LAG-3 is highly expressed on T_(regs) (CD4⁺CD25^(hi)) and mediates theirsuppressive function (Journal of immunology 184:6545-51, 2010). Toevaluate the ability of anti-LAG-3 antibodies on reversing thesuppressive effect of T_(regs) on effector T cells(CD4⁺CD25⁻CD127^(hi)), antibodies of Example 1 were used in an in vitrosuppression assay. First, T_(regs)(CD4⁺CD25^(hi)CD127^(low)) and Ten(CD4⁺CD25CD127^(hi)) were FACS-sorted by using a BD FACSAria II system.T_(effs) were then labeled with carboxyfluorescein succinimidyl ester(CFSE) and co-cultured with T_(regs) at a 1:1 ratio in the presence ofplate bound anti-CD3 antibodies and mitomycin C-treated antigenpresenting cells. Anti-LAG-3 antibodies were next added to the cellculture and T_(effs) cell proliferation were tested 5 days later. Theresults in FIG. 26, indicate that when Tregs were co-cultured witheffector T cells, effector T cell proliferation and cytokine productionwas inhibited. S119 and T99 can reverse the inhibition of T_(effs) byT_(regs).

2.7. LAG-3 antibody BIACORE Analysis

The binding of the S20, S24, S27, S31, S87, S119, S120, S128, S136, S161and T99 antibodies to recombinant his-tag human LAG3-ECD protein wasexamined by Biacore T200 using a capture method. Anti-LAG3 antibodieswere captured using anti-human Fc antibody. The anti-human Fc antibodywas coated on chip. Serial concentrations of his-tag human LAG3-ECDprotein (0-4 nM) were injected over capture antibodies at the flow rateof 30 μl/min. The dissociation phase was 900 s or 550 s. The results areshown in Table 22 below. The Biacore results for the anti-LAG3antibodies have shown that these anti-LAG3 antibodies are high affinitybinder to human LAG3.

TABLE 22 K_(a) (M⁻¹ s⁻¹) k_(d) (s⁻¹) K_(D) (M) S20 1.65E+05 7.33E−064.43E−11 S24 1.79E+06 1.20E−02 6.73E−09 S27 7.04E+06 1.10E−04 1.56E−11S31 2.08E+06 6.25E−05 3.00E−11 S87 9.28E+05 2.33E−06 2.51E−12 S1192.17E+07 1.49E−04 6.87E−12 S120 1.40E+06 2.64E−03 1.88E−09 S128 1.00E+068.17E−04 8.15E−10 S136 7.98E+05 8.27E−05 1.04E−10 S161 6.20E+05 5.53E−048.92E−10 T99 7.62E+06 1.70E−04 2.24E−11

2.8. Generation of Mouse Monoclonal Antibodies Against Human LAG3

This example shows how anti-human-LAG3 mouse monoclonal antibodies weregenerated using hybridoma technology.

Antigen: Recombinant human LAG-3 fusion proteins were used as theimmunogen to raise anti-human LAG-3 antibodies. A fusion proteincomprising the entire extracellular region (domains 1-4) of human LAG-3fused to a mouse immunoglobulin Fc domain (D1-D4 mFc) was used as theimmunogen. For the ELISA binding test, a fusion protein comprisingentire extracellular region (domains 1-4) or extracellular regionwithout D1-D2 domain of human LAG-3 fused to human immunoglobulin Fcdomain (D1-D4 huFc or ΔD1-D2 huFc respectively). The LAG-3 fusionproteins were prepared using standard recombinant DNA techniques.

Immunizations:

The LAG-3 fusion proteins were prepared using standard recombinant DNAtechniques. Mice were immunized intraperitoneally (IP) and/orsubcutaneously (SC). The mice were firstly SC immunized 50 mg immunogenand then IP immunized biweekly with 25 μg immunogen. The immune responsewas monitored by retroorbital bleeds. The plasma was screened by ELISAand cell-based receptor blocking assay (as described below). Mice withsufficient titers of anti-LAG-3 D1-D2 domain immunoglobulin andfunctional LAG3 blocker were used for fusions. Prior to sacrifice andremoval of the spleens, the mice were boosted intraperitoneally with 25μg of antigen followed by a subsequent boost with 25 μg of antigen. Thespleens were used for fusion. The hybridoma supernatant was tested foranti-LAG-3 D1-D2 domain binding and its function to block the binding ofLAG3 to its receptor by cell based receptor blocking assay.

Selection of Mice Producing Anti-LAG3 Blocking Antibodies.

To select mice producing anti-LAG3 blocking antibodies, sera fromimmunized mice was tested for binding to D1-D2 domain by ELISA. Briefly,sera were evaluated for their binding to D1-D4 huFc and its binding toΔD1-D2 huFc was served as a counter screen. In short, D1-D4 huFc orΔD1-D2 huFc was coated at 0.5 ug/ml overnight and then blocked by 5% BSAin PBS. The serially diluted sera were incubated with the coated antigenfor 1 h at room temperature. The resulting plates were washed with PBS/Tand incubated with goat anti-mouse IgG-HRP for 1 h at room temperature.The plates were developed with TMB substrate and analyzed byspectrophotometer at OD 450-630 nm. In parallel, sera were evaluated totheir function to blocking the binding of LAG3 to MHCII moleculesexpressed on Raji cells as described Example 2.4. The mice with hightiters specific to LAG3 D1-D2 domain and function to block the bindingof LAG3 to Raji cells were selected for fusion and further screening.

Hybridoma clones 122H, 147H and 170H were selected for further analysisand sequencing.

2.9. Binding Properties of Anti-LAG3 Mouse Monoclonal Antibodies

This example tested the binding properties of the anti-LAG3 mouseantibodies to the LAG3 proteins.

D1-D2 Specific Binders:

To evaluate the binding specificity, the purified 122H, 147H and 170Hmouse monoclonal antibodies were subjected to ELISA binding test forD1-D4 huFc and ΔD1-D2 huFc antigens. Briefly, D1-D4 huFc or ΔD1-D2 huFcwas coated at 0.5 μg/ml overnight and then blocked by 5% BSA in PBS. Theserially diluted antibodies (starting from 1 μg/ml and 1:3 serialdilution for 10 doses) were incubated with the coated antigen for 1 hrat room temperature. The resulting plates were washed with PBS/T andincubated with goat anti-mouse IgG-HRP for 1 h at room temperature. Theplates were developed with TMB substrate and analyzed byspectrophotometer at OD 450-630 nm.

The results of the ELISA are summarized in FIGS. 27A-27C, which showstrong binding to full extracellular domain of LAG3 (D1-D4 huFc) but notD1-D2 deleted LAG3 (ΔD1-D2 huFc), confirm that 122H, 147H and 170H arepotent and selective binder for D1 and D2 domain of human LAG3.

2.10. Functional Properties of Anti-LAG3 Mouse Monoclonal Antibodies

Blocking the Binding of LAG3 to its Receptor

To evaluate the ability of anti-LAG-3 antibodies to block the binding ofsLAG-3 to MHC class II receptor, an in vitro binding assay was designedusing biotin-labeled LAG-3-ECD-huFc fusion proteins and Raji cellsexpressing MHC class II receptor. 122H, 147H and 170H mouse monoclonalantibodies were serially diluted (1:5 for 6 doses) from 20 μg/mL withFACS buffer and pre-incubated with 6 ug/mL of biotin-LAG-3-ECD-huFc for30 min at room temperature. The antibody mixture was then added to FcRblocked Raji cells and incubated for 30 min on ice. Cells were thenwashed with FACS buffer and subsequently stained with streptavidin PEfor 30 min on ice and subsequently washed once with FACS buffer. Labeledcells were evaluated for fluorescence intensity by flow cytometry in aBD FACSCalibur™. As shown in FIGS. 28A-28C, the 122H, 147H and 170Hantibodies can dose dependently inhibit the binding of LAG3 to itsreceptor MHC class II molecules.

Stimulation of Human T Cell Response by Anti-LAG3 Antibodies

To test the ability of the anti-LAG3 antibodies to stimulated T cellresponse, Jurkat T cell stimulation assay was used. Jurkat is human Tcell leukemia cell line that can produce I12 upon TCR stimulation. Inthis assay, Jurkat cells transfected with human LAG3 gene by lentiviruswere used as the responder cells. The Raji cells which expressed MHCIIwas used as the antigen presenting cells (APC). StaphylococcalEnterotoxins (SE) are superantigen, which can crosslink the MHCIImolecules and T cell receptor beta (TCRVβ) and stimulate T cellresponse. SE was used as the stimulator in this assay. In this system,ectopically expressed huLAG3 can suppress SE stimulated IL-2 productionby Jurkat cells, while anti-LAG3 antibodies can reverse IL-2 production.In short, APCs (2.5×10⁴) were co-cultured with LAG3 expressing Jurkat Tcells (1×10⁵) in the presence of SE stimulation. Anti-LAG3 antibodies(starting from 20 μg/ml and 1:5 serially diluted for 6 dose) were addedat the beginning of the culture. 48 hr later, culture supernatant wasevaluated for IL2 production by ELISA. As shown in FIG. 29, 122H, 147Hand 170H mouse monoclonal antibodies can dose dependently promote IL2production by Jurkat T cells, suggesting they can stimulate TCRstimulation by suppressing LAG3 signal to T cells.

2.11. 147H Mouse mAb Humanization Design

The mAb 147H variable region genes were employed to create a humanizedmAb. In the first step of this process, the amino acid sequences of theVH and VK of mAb 147H were compared against the available database ofhuman Ig gene sequences to find the overall best-matching human germlineIg gene sequences. For the light chain, the closest human match was theA19/JK4 gene, and for the heavy chain the closest human match was theVH1-f/JH6 gene. Humanized variable domain sequences were then designedwhere the CDR1 (SEQ ID NO:243), 2 (SEQ ID NO:244) and 3 (SEQ ID NO:245)of the 147H light chain were grafted onto framework sequences of theA19/JK4 gene, and the CDR1 (SEQ ID NO:240), 2 (SEQ ID NO:241), and 3(SEQ ID NO:242) sequences of the 147H VH were grafted onto frameworksequences of the VH1-f/JH6 gene. A 3D model was then generated todetermine if there were any framework positions where replacing themouse amino acid to the human amino acid could affect binding and/or CDRconformation. In the case of the heavy chain, R71, M69, R66, V67, M48,V37, R38, Y91 and Q1 (Kabat numbering) in human framework wereidentified and subjected to back-mutation to their mouse counterpartamino acid i.e.: R71A, M69L, R66K, V67A, M481, V371, R38K, Y91F and Q1E.

TABLE 23 Mouse antibody sequences Antibody chain Sequences SEQ IDor domain (CDR residues with VH and VL are underlined) NO: 147H VHQVQLQQSGSELVRPGTSVKISCKASGYTFTNYWLGWIKQRPGHGLEWIG 352DIYPGGDYINYNEKFKGKATLSADTSSSTAYMQLSSLTSEDSAVYFCAR PNLPGDYWGQGTSVTVSS147H VL DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSP 353QLLIYQVSNLASGVPGRFSGSGSGTDFTLRISRVEAEDVGVYYCAQNLE LPWTFGGGTKLEIK CDRH1GYTFTNYWLG 354 CDRH2 DIYPGGDYINYNEKFKG 355 CDRH3 PNLPGDY 356 CDRL1RSSKSLLHSNGITYLY 357 CDRL2 QVSNLAS 358 CDRL3 AQNLELPWT 359

The amino acid sequences of the humanized antibodies are listed: 147H-1,147H-2, 147H-3, 147H-4, 147H-5, 147H-6, 147H-7, 147H-8, 147H-9, 147H-10,147H-11, 147H-12, 147H-13, and 147H-14, each having a different heavychain but all share a common light chain.

TABLE 24 Humanized antibodies and back mutations AntibodySequences (CDR underlined; back mutations bold and chain underlined)SEQ ID NO: 147H-1 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWVRQAPGQGLEWMGD360 IYPGGDYINYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARPN LPGDYWGQGTTVTVSS147H-2 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWVRQAPGQGLEWMGD 361IYPGGDYINYNEKFKGRVTMTADTSIST A YMELSRLRSDDTAVYYCARPN LPGDYWGQGTTVTVSS147H-3 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWVRQAPGQGLEWMGD 362IYPGGDYINYNEKFKGRVT L T A DTSISTAYMELSRLRSDDTAVYYCARPN LPGDYWGQGTTVTVSS147H-4 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWVRQAPGQGLEWMGD 363IYPGGDYINYNEKFKGKA T L T A DTSISTAYMELSRLRSDDTAVYYCARPN LPGDYWGQGTTVTVSS147H-5 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWVRQAPGQGLEW I GD 364IYPGGDYINYNEKFKGKA T L T A DTSISTAYMELSRLRSDDTAVYYCARPN LPGDYWGQGTTVTVSS147H-6 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGW IK QAPGQGLEW I G D 365IYPGGDYINYNEKFKGKA T L T A DTSISTAYMELSRLRSDDTAVYYCARPN LPGDYWGQGTTVTVSS147H-7 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGW IK QAPGQGLEW I GD 366IYPGGDYINYNEKFKGKA T L T A DTSISTAYMELSRLRSDDTAVY F CARPNLPGDYWGQGTTVTVSS 147H-8 VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWVRQAPGQGLEWMGD 367IYPGGDYINYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARPN LPGDYWGQGTTVTVSS147H-9 VH E VQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWVRQAPGQGLEWMGD 368IYPGGDYINYNEKFKGRVTMT A DTSISTAYMELSRLRSDDTAVYYCARPN LPGDYWGQGTTVTVSS147H-10 VH E VQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWVRQAPGQGLEWMGD 369IYPGGDYINYNEKFKGRVT L T A DTSISTAYMELSRLRSDDTAVYYCARPN LPGDYWGQGTTVTVSS147H-11 VH E VQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWVRQAPGQGLEWMGD 370IYPGGDYINYNEKFKGKA T L T A DTSISTAYMELSRLRSDDTAVYYCARPN LPGDYWGQGTTVTVSS147H-12 VH E VQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWVRQAPGQGLEW I GD 371IYPGGDYINYNEKFKGKA T L T A DTSISTAYMELSRLRSDDTAVYYCARPN LPGDYWGQGTTVTVSS147H-13 VH E VQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGW IK QAPGOGLEW I GD 372IYPGGDYINYNEKFKGKA T L T A DTSISTAYMELSRLRSDDTAVYYCARPN LPGDYWGQGTTVTVSS147H-14 VH E VQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGW IK QAPGQGLEW I GD 373IYPGGDYINYNEKFKGKA T L T A DTSISTAYMELSRLRSDDTAVY F CARPNLPGDYWGQGTTVTVSS 147H VLDIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQ 374LLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELP WTFGGGTKVEIK

The humanized VH and VK genes were produced synthetically and thenrespectively cloned into vectors containing the human gamma 1 and humankappa constant domains. The pairing of the human VH and the human VKcreated 40 humanized antibodies.

2.12. Binding Properties of Anti-LAG3 147H Humanized MonoclonalAntibodies

Affinity ranking of humanized antibodies by Octet® RED96 System

To explore the binding kinetics of the humanized antibody, this exampleperformed the affinity ranking by using Octet Red 96. As shown in Table25 below, 147H, 147H-6, 147H-7, 147H-13 and 147H-14 show betteraffinity.

TABLE 25 Antibody KD (M) kon(1/Ms) kdis(1/s) 147H-1 3.54E−08 1.09E+053.86E−03 147H-2 3.16E−08 9.93E+04 3.14E−03 147H-3 3.65E−08 9.25E+043.38E−03 147H-4 3.98E−08 8.62E+04 3.43E−03 147H-5 3.13E−08 9.58E+043.00E−03 147H-6 1.53E−08 1.20E+05 1.84E−03 147H-7 1.57E−08 1.52E+052.39E−03 147H-8 3.23E−08 1.65E+05 5.33E−03 147H-9 6.64E−08 6.74E+044.48E−03 147H-10 8.23E−08 4.91E+04 4.04E−03 147H-11 4.22E−08 1.07E+054.51E−03 147H-12 5.52E−08 6.23E+04 3.44E−03 147H-13 2.16E−08 1.08E+052.34E−03 147H-14 2.32E−08 1.08E+05 2.50E−03

Full kinetic affinity of humanized antibodies by Octet RED98 System

To explore the binding kinetics of the humanized antibody, this examplefurther performed the full kinetic affinity testing by running variousdose of antigen (50 nM, 25 nM, 12.5 nM, 6.15 nM, 3.125 nM) by usingOctet Red 96. The binding affinity was calculated by software in Octet®RED96 System. As shown in Table 26, 147H-6, 147H-7, 147H-13 and 147H-14showed comparable affinity with 147H chimeric antibody.

TABLE 26 Antibody KD (M) kon(1/Ms) kdis(1/s) 147H chimeric 2.71E−088.01E+04 2.17E−03 147H-6 2.48E−08 1.05E+05 2.59E−03 147H-7 2.65E−081.18E+05 3.12E−03 147H-13 1 82E−08 1.04E+05 1.90E−03 147H-14 2.07E−089.87E+04 2.04E−03

2.13. Functional Properties of Anti-LAG3 Mouse Monoclonal Antibodies

Stimulation of Human T Cell Response by Anti-LAG3 Antibodies

To test the ability of anti-LAG3 antibodies to stimulated T cellresponse, Jurkat T cell stimulation assay was used as described inExample 12. Anti-LAG3 antibodies (starting from 30 μg/ml and 1:3serially diluted for 6 doses) were added at the beginning of theculture. 48 hr later, culture supernatant was evaluated for IL2production by ELISA. As shown in FIG. 30, 147H-13 humanized monoclonalantibodies can dose dependently promote IL2 production by Jurkat Tcells, suggesting they can stimulate the TCR stimulation by suppressingLAG3 signal to T cells.

2.14. Affinity Maturation of Anti-LAG3 147H Humanized MonoclonalAntibodies

To improve antigen binding affinity, this example performed affinitymaturation of 147H4-13 using phage display technology. Strategy 1: TheCDRH3 and CDRL3 of 147H-13 were targeted for codon-based mutagenesis.CDRH3 and CDRL3 were randomized at position H95-H102 and L89-L97 (Kabatnumbering), respectively. Strategy 2: Each CDR was targeted for singlecodon based mutagenesis using CDR walking approach. Then CDRH1, CDRH2,CDRL1 combined to library 1. The CDRH3, CDRL2, CDRL3 combined to library2.

In both strategies, libraries were subject to three or four rounds ofaffinity-based solution-phase phage display selection with decreasingconcentration of antigen at each round. A relatively high antigenconcentration (10 nM) was used for the first round. The antigenconcentration was decreased 10-fold each of the subsequent three roundsor 100-fold each the subsequent two rounds to select for high affinityvariants. Individual variants from the final round were tested forpositive binding to antigen by ELISA screening. Off-rate ranking ofindividual variants was determined by Octet Red 96 (Fortebio, USA).Mutations with improved affinity were combined to generate new LAG3antibodies. Affinity was further confirmed by Biacore which suggestedN58V of CDR H2 significantly increased Koff, while N91Y of CDR L3improved Kon.

TABLE 27 Antibody affinity maturation No.Sequence (CDR underlined, mutation bold) 147H 3421 VH (SEQ ID NO: 375)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPKDH WGQGTTVTVSSVL (SEQ ID NO: 376)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3422VH (SEQ ID NO: 377)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADTSISTAYMEDSRLRSDDTAVYYCARPDLPGDYWGQGTTVTVSSVL (SEQ ID NO: 378)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISKVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3423VH (SEQ ID NO: 379)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPGLPKDYWGQGTTVTVSSVL (SEQ ID NO: 380)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3424VH (SEQ ID NO: 381)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADISISTAYMELSRLRSDDTAVYYCARPNLPKDYWGQGTTVTVSSVL (SEQ ID NO: 382)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3425VH (SEQ ID NO: 383)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPRDYWGQGTTVTVSSVL (SEQ ID NO: 384)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3426VH (SEQ ID NO: 385)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPGLPRDYWGQGTTVTVSSVL (SEQ ID NO: 386)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSROLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3427VH (SEQ ID NO: 387)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPGLPQDYWGQGTTVTVSSVL (SEQ ID NO: 388)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3428VH (SEQ ID NO: 389)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPDLPKDYWGQGTTVTVSSVL (SEQ ID NO: 390)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3429VH (SEQ ID NO: 391)EVQLVQSGAEVKKPGASVKVSOKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 392;DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDETLKISRVEAEDVGVYYC GQNLELPWTFGGGTKVEIK 147H 3430VH (SEQ ID NO: 393)EVQLVQSGAEVRRPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 394)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLEMPWTFGGGTKVEIK 147H 3431VH (SEQ ID NO: 395)EVQLVQSGAEVKKPGASVKVSCRASGYTFTNYWLGWIRQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 396)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC GQNLEMPWTFGGGTKVEIK 147H 3432VH (SEQ ID NO: 397)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYPWGQGTTVTVSSVL (SEQ ID NO: 398)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQYLEEPWTFGGGTKVEIK 147H 3433VH (SEQ ID NO: 399)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 400)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQYLELPWTFGGGTKVEIK 147H 3508VH (SEQ ID NO: 401)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPKDH WGQGTTVTVSSVL (SEQ ID NO: 402)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDETLKISRVEAEDVGVYYC GQNLELPWTFGGGTKVEIK 147H 3549VH (SEQ ID NO: 403)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPKDH WGQGTTVTVSSVL (SEQ ID NO: 404)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQYLEEPWTFGGGTKVEIK 147H 3550VH (SEQ ID NO: 405)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPKDH WGQGTTVTVSSVL (SEQ ID NO: 406)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQYLELPWTFGGGTKVEIK 147H 3663VH (SEQ ID NO: 407)EVQLVQSGAEVKKPGASVKVSCKASGYTFENYWLGWIKQAPGQGLEWIGDIYPGGDYIVYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 408)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLARGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3664VH (SEQ ID NO: 409)EVQLVQSGAEVKKPGASVKVSCKASGYMFTNYWLGWIKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 410)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQKSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3665VH (SEQ ID NO: 411)EVQLVQSGAEVKKPGASVKVSCKASGYTFDNYWLGWIKQAPGQGLEWIGDIYPGGDIINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 412)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLAVGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3666VH (SEQ ID NO: 413)EVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWLGWIKQAPGQGLEWIGDIYPGGDVINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVI (SEQ ID NO: 414)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLALGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3667VH (SEQ ID NO: 415) EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLWWIKQAPGQGLEWIGDIFPGGDYINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 416)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVDNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3668VH (SEQ ID NO: 417)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYIVYNEKFKGKATLTADTSISTTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 418)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLATGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3669VH (SEQ ID NO: 419)EVQLVQSGAEVKKPGASVKVSCKASGYLFTNYWLGWIKQAPGQGLEWIGDIYPGGDYIVYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 420)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDETLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3670VH (SEQ ID NO: 421)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 422)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIY HVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3675VH (SEQ ID NO: 423) EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLWWIKQAPGQGLEWIGDIYPGGDLINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 424)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIY HVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3676VH (SEQ ID NO: 425) EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLSWIKQAPGQGLEWIGDIYPGGDHINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 426)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3677VH (SEQ ID NO: 427) EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLW WIKQAPGQGLEWIGEIYPGGDYITYN EKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 428)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3678VH (SEQ ID NO: 429)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADISISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 430)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVDNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3679VH (SEQ ID NO: 431)EVQLVQSGAEVKKPGASVKVSCKASGFTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYIVYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 432)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3790VH (SEQ ID NO: 433)EVQLVQSGAEVRRPGASVKVSCKASGYTTNYWLGWLKQAPGQGLEWIGDIYPGGDYINYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPKDH WGQGTTVTVSSVL (SEQ ID NO: 434)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLATGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEIK 147H 3791VH (SEQ ID NO: 435)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYIVYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 436)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDETLKISRVEAEDVGVYYC GQNLELPWTFGGGTKVEIK 147H 3792VH (SEQ ID NO: 437)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYIVYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 438)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDETLKISKVEAEDVGVYYCAQYLELPWTFGGGTKVEIK 147H 3793VH (SEQ ID NO: 439)EVQLVQSGAEVKKPGASVKVSCKASGYLFTNYWLGWIKQAPGQGLEWIGDIYPGGDYIVYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 440)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC GQNLELPWTFGGGTKVEIK 147H 3794VH (SEQ ID NO: 441)EVQLVQSGAEVKKPGASVKVSCKASGYLFTNYWLGWIKQAPGQGLEWIGDIYPGGDYIVYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPGDYWGQGTTVTVSSVL (SEQ ID NO: 442)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQYLELPWTFGGGTKVEIK 147H 3807VH (SEQ ID NO: 443)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYIVYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPKDH WGQGTTVTVSSVL (SEQ ID NO: 444)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSQGITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQYLELPWTFGGGTKVEIK 147H 3807bVH (SEQ ID NO: 491)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYIVYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPKDH WGQGTTVTVSSVL (SEQ ID NO: 492)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQYLELPWTFGGGTKVEIK 147H 3808VH (SEQ ID NO: 445)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWIKQAPGQGLEWIGDIYPGGDYIVYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPKDH WGQGTTVTVSSVL (SEQ ID NO: 446)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC GQYLELPWTFGGGTKVEIK 147H 3809VH (SEQ ID NO: 447)EVQLVQSGAEVKKPGASVKVSCKASGYLFTNYWLGWIKQAPGQGLEWIGDIYPGGDYIVYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPKDH WGQGTTVTVSSVL (SEQ ID NO: 448)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQYLELPWTFGGGTKVEIK 147H 3810VH (SEQ ID NO: 449)EVQLVQSGAEVKKPGASVKVSCKASGYLFTNYWLGWIKQAPGQGLEWIGDIYPGGDYIVYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPKDH WGQGTTVTVSSVL (SEQ ID NO: 450)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLATGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQYLELPWTFGGGTKVEIK 147H 3811VH (SEQ ID NO: 451)EVQLVQSGAEVKKPGASVKVSCKASGYLFTNYWLGWIKQAPGQGLEWIGDIYPGGDYIVYNEKFKGKATLTADTSISTAYMELSRLRSDDTAVYYCARPNLPKDH WGQGTTVTVSSVL (SEQ ID NO: 452)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNAITYLYWYLQKPGQSPQLLIYQVSNLATGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC GQYLELPWTFGGGTKVEIK

TABLE 28 Summary of mutations and mutated CDR regions: Examplesubstitutions Original sequence (based on kabatExample mutated sequences (SEQ ID NO:_) numbering) (SEQ ID NO:_) CDRH1 GYT F T NYWL G  (354 ) Y27: F GYTF E NYWLG (453) T28: M, L GY MFTNYWLG (454) T30: E, D, G GYTF D NYWLG (455) 527: W, S GYTF GNYWLG (456) GYTFTNYWL W  (457) GY L FTNYWLG (458) GYTFTNYWL S  (459) G FTFTNYWLG (460) CDRH2 D I Y PGGD Y I N YNEKFKG D50: E DIYPGGDYI VYNEKFKG (461) (355) Y52: F DIYPGGD I INYNEKFKG (462) Y56: 1, V, L, HDIYPGGD V INYNEKFKG (463) N58: V, T DI F PGGDYINYNERHKG (464) DIYPGGD LINYNEKFKG (465) DIYPGGD H INYNEKEKG (466) E IYPGGDYI T YNEKFKG (467)CDRH3 P N LP G D Y  (356) N96: D, G PNLP K D H  (468) G99: K, R, Q P DLPGDY (469) Y102: H P G LP K DY (470) PNLP K DY (471) PNLP R DY (472) PG LP R DY (473) P G LP Q DY (474) P D LP K DY (475) CDRL1 RSSKSLLHS NGITYLY N28: Q RSSKSLLHS Q GITYLY (490) (357) CDRL2 QVS N L A S  (358)Q50: H QVSNLA R  (476) V51: K Q K SNLAS (477) S52: D QVSNLA V  (478)L54: R QVSNLA L  (479) S56: R, V, L, T QV D NLAS (480) QVSNLA T  (481) HVSNLAS (482) QVSN R AS (483) CDRL3 A Q N LE L PWT (359) A89: G GQNLELPWT (484) N91: Y AQNLE M PWT (485) L94: M, E G QNLE M PWT (486) AQY LE E PWT (487) AQ Y LELPWT (488) G Q Y LELPWT (489)

2.15. Binding Properties of Affinity Matured Anti-LAG3 147H HumanizedMonoclonal Antibodies

The binding kinetics of affinity matured antibodies to recombinanthis-tag human LAG3-ECD protein was examined by Biacore T200, as statedin Example 2.7. The results were shown in Table below. The Biacoreresults showed that these anti-LAG3 antibodies had better affinity thanparent 147H-13.

TABLE 29 KD (M) kon(1/Ms) kdis(1/s) 147H-13 1.4E−08 2.2E+06 3.0E−02 147H3421 8.1E−09 1.4E+06 1.2E−02 147H 3508 1.4E−09 2.9E+06 4.2E−03 147H 35499.2E−10 7.4E+06 6.8E−03 147H 3550 9.8E−10 8.7E+06 8.5E−03 147H 36636.8E−09 7.9E+05 5.4E−03 147H 3669 8.8E−09 7.2E+05 6.3E−03 147H 37905.9E−09 7.7E+05 4.5E−03 147H 3791 1.2E−09 2.1E+06 2.5E−03 147H 37925.9E−10 4.9E+06 2.9E−03 147H 3793 1.3E−09 1.8E+06 2.3E−03 147H 37947.2E−10 3.7E+06 2.7E−03 147H 3807b 5.1E−10 4.0E+06 2.0E−03 147H 38087.5E−10 4.3E+06 3.2E−03 147H 3809 4.7E−10 4.3E+06 2.0E−03 147H 38104.1E−10 4.7E+06 1.9E−03 147H 3811 5.9E−10 4.9E+06 2.9E−03

To confirm the capability of affinity matured anti-LAG-3 antibodiesbinding to human LAG3, 2 antibodies with highest affinity (B3807b andB3810) along with parent antibody 147H-13 were evaluated using ELISA,which was described in Example 2.2. EC50 of B3807b, B3810 along withparent antibody was showed in table below. Both B3807b and B3810 showedsuperior binding capability than parent antibody 147H-13.

TABLE 30 Name EC50 (nM) 147H-13 6.5 147H 3807b 0.41 147H 3810 0.49

To further confirm affinity matured anti-LAG-3 antibodies could bind tocell-derived human LAG3, both inducible hLAG3 expressed Jurkat cells andactivated PBMCs were used to test the binding capability of B3807b andB3810. In brief, Jurkat cells were resuspended in FACS buffer. Anti-LAG3antibodies and isotype control were 4-fold serially diluted in FACSbuffer with a dose ranging from 20 nM to 30 pM. The serially dilutedantibodies were added to the cell suspension and incubated for 30minutes on ice. Then after removal of unbound antibodies, cells werestained with anti-human IgG conjugated with Alexa Fluor 633 (Thermo,A21091). Fluorescence measurement was acquired on FACSCelesta flowcytometer and analyzed in Flowjo to determine the mean fluorescenceintensities (MFI). To test anti-LAG3 antibodies' ability of binding tonative human LAG3, PBMCs from health donor were stimulated with anti-CD3(BD, 555336) and anti-CD28 (BD, 555725) both at a concentration of 1ug/ml. Following 3 days' stimulation, cells were harvested and incubatedwith anti-LAG3 antibodies for 30 mins on ice. The cells were stainedwith anti-human CD4 and anti-human IgG. Analysis of antibodies bindingto CD4+ cells were carried out on FACSCelesta flow cytometry. Theresults of cytometry analysis were summarized in table below whichshowed EC50 of antibodies binding to cell-derived human LAG3. FIG. 31 isa graph showing the binding curve of anti-LAG3 antibodies. EC50 oftested antibodies was showed below.

TABLE 31 EC50 (nM) Cell-based binding assay 147H-13 147H 3807b 147H 3810Jurkat-LAG3 1.2 0.4 0.5 Activated CD4 T cells 0.77 0.33 0.39

2.16. Characterization of Monoclonal Antibody 147H 3807 (B3807)

A. Binding of B3807 to LAG3 Protein

This example evaluated the capability of the anti-LAG-3 antibody 147H3807 (B3807) to bind to the human LAG3 protein. The streptavidin wascoated to an ELISA plate at 2 μg/ml with 100 μl/well. 100 μl of Bio-LAG3at 1.0 μg/ml was subsequently incubated with streptavidin at RT for 1hr. B3807, along with a positive control 25F7 and a negative controlIgG, were serially diluted with ELISA diluent buffer. To assess binding,the antibodies at various concentrations were added to LAG3protein-coated plate for 1.5 hr RT. The resulting plates were washed andthen labeled with anti-human IgG(Fab)-HRP antibody.

As shown in FIG. 38, both B3807 and 25F7 bound to human LAG3 in adose-dependent manner, with B3807 showing a higher potency and lowerEC50 (0.06 nM vs. 0.22 nM for 25F7).

B. Biacore Analysis

The binding of B3807 to recombinant His-tag human LAG3-ECD protein wasexamined by Biacore T200 using a capture method. B3807 was capturedusing protein A which was immobilized on CM5 sensor chip. Serialconcentrations of his-tag human LAG3-ECD protein (0-12 nM) were injectedover capture antibodies at the flow rate of 30 μl/min. The dissociationphase was 900 s or 550 s. The results are shown in FIG. 39,demonstrating that B3807 is binding to human LAG3 with high affinity

C. Jurkat Cell and PBMC-Based Binding Assays

To further confirm that B3807 could bind to cell-derived human LAG3,both inducible human LAG3 expressed Jurkat cells and activated PBMCswere used to test the binding capability of B3807. In brief, Jurkatcells were resuspended in FACS buffer. B3807, 25F7 and isotype controlwere 3-fold serially diluted in FACS buffer with a dose ranging from 20nM to 9 pM. The serially diluted antibodies were added to the cellsuspension and incubated for 30 minutes on ice. Then after removal ofunbound antibodies, cells were stained with anti-human IgG conjugatedwith Alexa Fluor 633 (Thermo, A21091). Fluorescence measurement wasacquired on FACSCelesta flow cytometer and analyzed in Flowjo todetermine the mean fluorescence intensities (MFI). To test theantibodies' ability of binding to native human LAG3, PBMCs from healthdonor were stimulated with anti-CD3 (BD, 555336) and anti-CD28 (BD,555725) both at a concentration of 1 μg/ml. Following 3 days'stimulation, cells were harvested and incubated with anti-LAG3antibodies for 30 mins on ice. The cells were stained with anti-humanCD4 and anti-human IgG. Analysis of antibodies binding to CD4+ cellswere carried out on FACSCelesta flow cytometry.

The results of cytometry analysis are presented in FIG. 40. EC50 oftested antibodies are also showed in the figure. In both tests, B3807exhibited stronger binding capability than the control antibody 25F7.

D. Blocking of LAG3 Binding to MHC Class II

To measure the ability of B3807 to block the interaction between humanLAG3 and MHCII, the LAG3 and MHC II binding assay (Cisbio, 64ICP03PEG)was performed utilizing homogeneous TR-FRET technology, following theprotocol provided by the kit manufacturer. B3807 was 3-fold dilutedranging from 100 nM to 5 pM (10 points). Fluorescence data was acquiredon a PerkinElmer Envision plate reader and a four-parameterdose-response curve was fitted to obtain IC50 of each antibody. IC50 ofB3807 was 0.41 nM (FIG. 41) demonstrating potent blocking activity.

E. Stimulation of Human T Cell Response

To test the ability of anti-LAG3 antibodies to stimulate T cellresponse, hLAG3-expressed Jurkat cells were used. In each well of96-well plate, Jurkat cells (1×10⁵) were incubated with Raji cells(1×10⁴) in the presence of 0.1 ng/ml SE. B3807 was 3-fold diluted andadded to the cells at a final concentration ranging from 100 nM to 50pm. 48 hours later, IL2 from the culture medium was measured using ahomogeneous TR-FRET assay (PerkinElmer, TRF1221M). FIG. 42 shows thecurve of B3807 and 25F7 in stimulating IL2 release, in which B3807outperformed 25F7 by a great margin.

F. IL2 Release in Primary T Cells

The antibodies' ability to stimulate T cell response was also testedwith hLAG3-expressed primary T cells. At all four tested doses, B3807outperformed 25F7 (FIG. 43, left panel). When used with an anti-PD-L1antibody together, the IL2 release profile (FIG. 43, right panel)demonstrated the synergistic effect between the anti-LAG3 antibody B3807and the anti-PD-L1 antibody.

G. Combinatory Effects with Anti-PD1/Anti-PD-L1 Antibodies in TumorRegression

Humanized mice that expressed the extracellular domains of human LAG3were used. As shown in FIG. 44, left panel, B3807 and 25F7 exhibitedsome effect in inhibiting the tumor growth when combo with anti-PD-1antibody.

In the right panel of FIG. 44, however, it is apparent that both B3807and 25F7 had significant synergistic effect when used together withTecentriq, a commercially available anti-PD-L1 antibody.

H. Comparison of B3807 with B3807b

The activities of B3807 and B3807b were compared for their ability inpromoting IL2 release in Jurkat cells (see experimental procedure inExample 2.16(E)) and in binding to LAG3 on Jurkat cells (seeexperimental procedure in Example 2.16(C)).

The comparison results are presented in FIG. 45. In both experiments,B3807 and B3807b exhibited highly similar activity profiles,demonstrating that the sequence difference in CDRL1 between these twoantibodies did not impact their activities.

Also, as shown in FIG. 46, the Biacore data (see experimental procedurein Example 2.16(B)) further demonstrate that the great similaritybetween these two antibodies. B3807 was used in the following examplesfor further testing and preparing bispecific antibodies.

Example 3. Preparation of Anti-PD-L1/Anti-LAG3 Bispecific Antibodies

Hu1210-41 (Hu1210 VH.4dxHu1210 Vk.1, see Table 8; hereinafter, “H12”)and B6 (see Table 16) clones among the anti-PD-L1 clones prepared inExample 1 and 147H (also called as “147”, see Table 23) and 147H 3807(also called as “147(H3807)”; see Table 27) clones among the anti-LAG3clones prepared in Example 2 were exemplarily selected, to prepareanti-PD-L1/anti-LAG3 bispecific antibodies in a full-length IgG X scFvform. When PD-L1 is placed in full IgG part, IgG1 with ADCC reducedmutant backbone (N297A mutation; U.S. Pat. Nos. 7,332,581, 8,219,149,etc.) was used, and when LAG3 is placed in full IgG part, IgG4 was usedwith S241P mutation (Angal et al., Mol. Immunol. 30:105-108).

A DNA segment 1 having a nucleotide sequence encoding a heavy chain ofan IgG antibody of the anti-PD-L1/anti-LAG3 bispecific antibody wasinserted into pcDNA 3.4 (Invitrogen, A14697; plasmid 1), and a DNAsegment 2 having a nucleotide sequence encoding a light chain of an IgGantibody of the anti-PD-L1/anti-LAG3 bispecific antibody was insertedinto pcDNA 3.4 (Invitrogen, A14697; plasmid 2). Thereafter, a DNAsegment 3 encoding a scFv was fused at a part of the DNA segment 1corresponding to the c-terminus of the Fc region of the IgG antibodyinserted into the plasmid 1, using a DNA segment 4 encoding a linkerpeptide having 10 amino acid lengths consisting of (GGGGS)2, toconstruct vectors for the expression of bispecific antibodies.

The sequences of the heavy chain, light chain, scFv and DNA segmentswere summarized in Tables 32 and 33:

TABLE 32Bispecific antibody comprising the anti-PD-L1 clone in IgG form andthe anti-LAG3 clone in scFvform (PD-L1xLAG3) Amino acid sequence (N′→C′)Nucleotide Sequence (5′→3′) H12x147(bispecific antibody comprising the anti-PD-L1 H12 done in IgG formand the anti-LAG3 147 clone in scFv form) Heavy Heavy chain ofEVQLVESGGGLVQPG GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTG Chain H12GSLRLSCAASGFTFS GTGCAACCCGGAGGCAGCCTGAGACTGAGCTGC SYDMSWVRQAPGKSLGCTGCCAGCGGCTTCACCTTCAGCAGCTACGAC EWVATISDAGGYIYYATGAGCTGGGTGAGACAGGCCCCTGGCAAAAGC SDSVKGRETISRDNACTGGAGTGGGTGGCCACCATCTCCGATGCGGGC KNSLYLQMNSLRDEDGGCTACATCTATTACTCCGACAGCGTGAYGGGC TAVYICAREFGKRYAAGGTTCACCATCAGCAGGGACAACGCCAAGAAC LDYWGQGTTVTVSSAAGCCTGTACCTGCAGATGAACAGCCTGAGGGAT STKGPSVFPLAPSSKGAGGACACCGCCGTGTACATCTGCGCCAGGGAG STSGGTAALGCLVKDTTCGGCAAAAGGTACGCCCTGGACTACTGGGGC YFPEPVTVSWNSGALCAGGHCACAACCGTGACCGTGAGCAGCgctAgc TSGVHTFPAVLQSSGAccAAgGGCCCCTCTGTGTTCCCTCTGGCCCCT LYSLSSVVTVPSSSLTCCTCTAAATCCACCTCTGGCGGAACCGCTGCT GTQTYICNVNHKPSNCTGGGCTGTCTGGTCAAGGACTACTTCCCTGAG TKVDKKVEPKSCDKTCCCGTGACCGTGTCTTGGAATTCTGGCGCTCTG HTCPPCPAPELLGGPACCAGCGGAGTGCACACCTTTCCAGCTGTGCTG SVFLFPPKPKDTLMICAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTC SRTPEVTCVVVDVSHGTGACAGTGCCTTCCAGCTCTCTGGGCACCCAG EDPEVKFNWYVDGVEACCTACATCTGCAACGTGAACCACAAGCCCTCC VHNAKTKPREEQYASAACACCAAGGTGGACAAGAAGGTGGAACCCAAG TYRVVSVLTVLHQDWTCCTGCGACAAGACCCACACCTGTCCTCCATGT LNGKEYKCKVSNKALCCTGCTCCAGAACTGCTGGGCGGACCCTCCGTG PAPIEKTISKAKGQPTTCCTGTTCCCTCCAAAGCCTAAGGACACCCTG REPQVYTLPPSREEMATGATCTCCCGGACCCCTGAAGTGACCTGCGTG TKNQVSLTCLVKGFYGTGGTGGATGTGTCCCACGAGGATCCCGAAGTG PSDIAVEWESNGQPEAAGTTCAATTGGTACGTGGACGGCGTGGAAGTG NNYKTTPPVLDSDGSCACAACGCCAAGACCAAGCCTAGAGAGGAACAG FFLYSKLTVDKSRWQTACgccTCCACCTACCGGGTGGTGTCCGTGCTG QGNVFSCSVMHEALHACCGTTCTGCACCAGGATTGGCTGAACGGCAAA NHYTQKSLSLSPGKGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTG (SEQ ID NO: 528)CCTGCCCCTATCGAAAAGACCATCTCTAAGGCC Linker GSGSGSGSGSGSGSGAAGGGCCAGCCCCGGGAACCTCAAGTGTACACC SGS TTGCCTCCCAGCCGGGAAGAGATGACCAAGAAC(SEQ ID NO: 529) CAGGTGTCCCTGACCTGCCTGGTTAAGGGCTTC scFv of VLDIVMTQSPLSLPVTP TACCCCTCCGATATCGCCGTGGAATGGGAGTCT 147 GEPASISCRSSKSLLAATGGCCAGCCTGAGAACAACTACAAGACCACA HSNGITYLYWYLQKPCCTCCTGTGCTGGACTCCGACGGCTCATTCTTC GQSPQLLIYQVSNLACTGTACTCCAAGCTGACCGTGGACAAGTCCAGA SGVPDRFSGSGSGTDTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTG FTLKISRVEAEDVGVATGCACGAGGCCCTGCACAATCACTACACCCAG YYCAQNLELPWTFGCAAGTCCCTGTCTCTGTCCCCTGGCAAAGGCTCC GTKVEIKRGGATCTGGTTCTGGATCCGGAAGCGGTTCTGGC (SEQ ID NO: 530)AGCGGCTCTGGATCTGACATCGTGATGACCCAG Linker GGGGSGGGSGGGGSTCTCCACTGAGCCTGCCTGTGACACCTGGCGAG GGGGSCCTGCTTCCATCTCCTGCCGGTCCTCTAAGTCC (SEQ ID NO: 531)CTGCTGCACTCTAACGGCATCACCTACCTGTAC VH EVQLVQSGAEVKKPGTGGTATCTGCAGAAGCCCGGCCAGTCTCCTCAG ASVKVSCKASGYTFTCTGCTGATCTACCAGGTGTCCAACCTGGCTTCT NYWLGWIKQAPGQCLGGCGTGCCCGATAGATTCTCCGGTAGCGGATCT EWIGDIYPGGDYINYGGAACCGACTTCACCCTGAAGATCTCGAGAGTG NEKFKGKATLTADTSGAAGCCGAGGACGTGGGCGTGTACTACTGTGCC ISTAYMELSRLRSDDCAGAACCTGGAACTGCCCTGGACCTTTGGCTGT TAVYYCARPNLPGDYGGCACCAAGGTGGAAATCAAGAGAGGCGGCGGA WGQGTTVTVSS*GGATCTGGCGGAGGTGGAAGCGGAGGCGGAGGA (SEQ ID NO: 532)AGCGGTGGCGGCGGATCTGAAGTTCAGTTGGTT CAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCTTCCGGCTAC ACCTTTACCAACTACTGGCTCGGCTGGATCAAGCAGGCCCCTGGACAGTGTCTGGAATGGATCGGC GACATCTACCCTGGCGGCGACTACATCAACTACAACGAGAAGTTCAAGGGCAAAGCTAGCCTGACC GCCGACACCTCTATCTCCACCGCCTACATGGAACTGTCCCGGCTGAGATCTGACGACACCGCCGTG TACTATTGCGCCAGACCTAACCTGCCTGGCGACTATTGGGGCCAGGGCACAACAGTGACCGTGTCC TCTTAA (SEQ ID NO: 533) LightLight chain of DIQMTQSPSSLSASV GACATCCAGATGACCCAGAGCCCTAGCAGCCTG chainH12 GDRVTITCKASQDVT AGCGCTAGCGTGGGCGACAGGGTGACCATCACC PAVAWYQQKPGKAPKTGCAAGGCCAGCCAGGATGTGACCCCTGCCGTG LLIYSTSSRYTGVPSGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCC RFSGSGSGTDFTETIAAGCTGCTGATCTACAGCACCAGCAGCAGGTAC SSLQPEDIATYYCQQACCGGCGTGCCCAGCAGGTTTAGCGGAAGCGGC HYTTPLTFGQGTKLEAGCGGCACCGACTTCACCTTCACCATGAGCAGC IKRTVAAPSVFIFPPCTGCAGCCCGAGGACATCGCCACCTACTACTGC SDEQLKSGTASVVCLCAGCAGCACTACACCACCCCTCTGACCTTCGGC LNNEYPREAKVOWKVCAGGGCACCAAGCTGGAGATCAAGAGAACCGTG DNALQSGNSQESVTEGCCGCTCCCTCCGTGTTCATCTTCCCACCATCT QDSKDSTYSLSSTLTGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTC LSKADYEKHKVYACEGTGTGCCTGCTGAACAACTTCTACCCTCGGGAA VTHQGLSSPVTKSFNGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTG RGEC*CAGTCCGGCAACTCCCAAGAGTCTGTGACCGAG (SEQ ID NO: 534)CAGGACTCCAAGGACAGCACCTACTCCCTGTCC TCTACCCTGACCCTGTCCAAGGCCGACTAGGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCAC CAGGGACTGTCTAGCCCCGTGACCAAGTCCTTCAACAGAGGCGAGTGCTGA (SEQ ID NO: 535) H12x147(H3807)(bispecific antibody comprising the anti-PD-L1 H12 clone in IgG formand the anti-LAG3 147(H3807) done in scFv form) Heavy Heavy chain ofEVQLVESGGGLVQPG GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTG Chain H12GSLRLSCAASGFTFS GTGCAACCCGGAGGCAGCCTGAGACTGAGCTGC SYDMSWVRQAPGKSLGCTGCCAGCGGCTTCACCTTCAGCAGCTACGAC EWVATISDAGGYIYYATGAGCTGGGTGAGACAGGCCCCTGGCAAAAGC SDSVKGRFTISRDNACTGGAGTGGGTGGCCACCATCTCCGATGCGGGC KNSLYLQMNSLRDELGGCTACATCTATTACTCCGACAGCGTGAAGGGC TAVYICAREFGKRYAAGGTTCACCATCAGCAGGGACAACGCCAAGAAC LDYWGQGTTVTVSSAAGCCTGTACCTGCAGATGAACAGCCTGAGGGAT STKGPSVFPLAPSSKGAGGACACCGCCGTGTACATCTGCGCCAGGGAG STSGGTAALGCLVKDTTCGGCAAAAGGTACGCCCTGGACTACTGGGGC YFPEPVTVSWNSGALCAGGGCACAACCGTGACCGTGAGCAGCgctAgc TSGVHTFPAVLQSSGAccAAgGGCCCCTCTGTGTTCCCTCTGGCCCCT LYSLSSVVTVPSSSLTCCTCTAAATCCACCTCTGGCGGAACCGCTGCT GTQTYICNVNHKPSNCTGGGCTGTCTGGTCAAGGACTACTTCCCTGAG TKVDKKVEPKSCDKTCCCGTGACCGTGTCTTGGAATTCTGGCGCTCTG HTCPPCPAPELLGGPACCAGCGGAGTGCACACCTTTCCAGCTGTGCTG SVFLFPPKPKDTLMICAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTC SRTPEVTCVVVDVSHGTGACAGTGCCTTCCAGCTCTCTGGGCACCCAG EDPEVKFNWYVDGVEACCTACATCTGCAACGTGAACCACAAGCCCTCC VHNAKTKPREEQYASAACACCAAGGTGGACAAGAAGGTGGAACCCAAG TYRVVSVLTVLHQDWTCCTGCGACAAGACCCACACCTGTCCTCCATGT LNGKEYKCKVSNKALCCTGCTCCAGAACTGCTGGGCGGACCCTCCGTG PAPIEKTISKAKGQPTTCCTGTTCCCTCCAAAGCCTAAGGACACCCTG REPQVYTLPPSREEMATGATCTCCCGGACCCCTGAAGTGACCTGCGTG TKNQVSLTCLVKGFYGTGGTGGATGTGTCCCACGAGGATCCCGAAGTG PSDIAVEWESNGQPEAAGTTCAATTGGTACGTGGACGGCGTGGAAGTG NNYKTTPPVLDSDGSCACAACGCCAAGACCAAGCCTAGAGAGGAAGAG FFLYSKLTVDKSRWQTACgccTCCACCTACCGGGTGGTGTCCGTGCTG QGNVFSCSVMHEALHACCGTTCTGCACCAGGATTGGCTGAACGGCAAA NHYTQKSLSLSPGKGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTG (SEQ ID NO: 528)CCTGCCCCTATCGAAAAGACCATCTCTAAGGCC Linker GSGSGSGSGSGSGSGAAGGGCCAGCCCCGGGAACCTCAAGTGTACACC SGS TTGCCTCCCAGCCGGGAAGAGATGACCAAGAAC(SEQ ID NO: 529) CAGGTGTCCCTGACCTGCCTGGTTAAGGGCTTC scFv of VLDIVMTQSPLSLPVTP TACCCCTCCGATATCGCCGTGGAATGGGAGTCT 147(H3807)GEPASISCRSSKSLL AACGGCCAGCCCGAGAACAACTACAAGACCACC HSQGITYLYWYLQKPCCTCCTGTGCTGGACTCCGACGGCTCATTCTTC GQSPQLLIYQVSNLACTGTACTCCAAGCTGACCGTGGACAAGTCTCGG SGVPDRFSGSGSGTDTGGCAGCAGGGCAACGTGTTCTCCTGCTCTGTG FTLKISRVEAEDVGVATGCACGAGGCCCTGCACAACCACTACACCCAG YYCAQYLELPWTFGCGTGCAACCCGGAGGCAGCCTGAGACTGAGCTGC GTKVEIKRAAGTCCCTGTCCCTGTCTCCCGGCAAAGGCTCC (SEQ ID NO: 536)GGATCTGGTTCTGGATCCGGAAGCGGTTCTGGC Linker GGGGSGGGGSGGGGSAGCGGCTCTGGATCTGACATTGTGATGACCCAG GGGGSAGCCCCCTGAGCCTCCCCGTGACCCCTGGAGAA (SEQ ID NO: 531)CCCGCCAGCATAAGCTGCAGATCCTCCAAAAGC VH EVQLVQSGAEVKKPGCTGCTGCACTCCCAGGGAATAACCTACCTGTAT ASVKVSCKASGYTFTTGGTACCTGCAGAAACCCGGCCAATCCCCCCAA NYWLGWIKQAPGQCLCTCCTGATATACCAAGTGTCCAACCTGGCCTCC EWIGDIYPGGDYIVYGGCGTGCCCGACAGATTCTCCGGCTCCGGCAGC NEKFKGKATLTADTSGGTACCGACTTCACCCTCAAAATCTCCAGAGTG ISTAYMELSRLRSDDGAAGCAGAAGACGTCGGCGTGTACTACTGCGCC TAVYYCARPNLPKDRCAGTACCTGGAACTGCCCTGGACCTTCGGCtgt WGQGTTVTVSS*GGCACCAAGGTGGAAATCAAGAGAGGCGGCGGA (SEQ ID NO: 537)GGAAGCGGAGGCGGCGGTTCTGGTGGTGGCGGT AGCGGAGGTGGTGGATCTGAGGTGCAGCTGGTGCAGAGCGGAGCAGAGGTGAAGAAGCCAGGGGCC AGCGTGAAGGTGAGCTGTAAGGCTAGTGGGTACACATTTACAAACTATTGGCTGGGATGGATTAAG CAGGCCCCAGGCCAAtgcCTGGAGTGGATAGGAGACATATACCCCGGAGGAGACTATATCGTGTAC AACGAGAAGTTCAAGGGCAAGGCCACACTCACCGCTGATACAAGCATCAGCACCGCCTACATGGAG CTGAGCCGACTGAGAAGCGACGACACAGCAGTGTATTACTGCGCCAGACCCAACCTGCCCAAGGAC CACTGGGGACAAGGCACCACCGTGACCGTGAGCAGCtga (SEQ ID NO: 538) Light Light chain of DIQMTQSPSSLSASVGACATCCAGATGACCCAGAGCCCTAGCAGCCTG chain H12 GDRVTITCKASQDVTAGCGCTAGCGTGGGCGACAGGGTGACCATCACC PAVAWYQQKPGKAPKTGCAAGGCCAGCCAGGATGTGACCCCTGCCGTG LLIYSTSSRYTGVPSGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCC RFSGSGSGTDFTFTIAAGCTGCTGATCTACAGCACCAGCAGCAGGTAC SSLQPEDIATYYCQQACCGGCGTGCCCAGCAGGTTTAGCGGAAGCGGC HYTTPLTFGQGTKLEAGCGGCACCGACTTCACCTTCACCATCAGCAGC IKRTVAAPSVFIFPPCTGCAGCCCGAGGACATCGCCACCTACTACTGC SDEQLKSGTASVVCLCAGCAGCACTACACCACCCCTCTGACCTTCGGC LNNEYPREAKVQWKVCAGGGCACCAAGCTGGAGATCAAGAGAACCGTG DNALQSGNSQESVTEGCCGCTCCCTCCGTGTTCATCTTCCCACCATCT QDSKDSTYSLSSTLTGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTC LSKADYEKHKVYACEGTGTGCCTGCTGAACAACTTCTACCCTCGGGAA VTHQGLSSPVTKSFNGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTG RGEC*CAGTCCGGCAACTCCCAAGAGTCTGTGACCGAG (SEQ ID NO: 534)CAGGACTCCAAGGACAGCACCTACTCCCTGTCC TCTACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCAC CAGGGACTGTCTAGCCCCGTGACCAAGTCCTTCAACAGAGGCGAGTGCTGA (SEQ ID NO:535) B6x147(bispecific antibody comprising the anti-PD-L1 B6 clone in IgG formand the anti-LAG3 147 clone in scFv form) Heavy Heavy chain ofEVQLVESGGGLVQPG  GAAGTGCAGCTGGTTGAATCTGGCGGCGGATTG Chain B6GSLRLSCAASGFTFS GTTCAGCCTGGCGGATCTCTGAGACTGTCTTGT SYDMSWVRQAPGKSLGCCGCCTCCGGCTTCACCTTCTCCAGCTACGAT EWVATISDAGGYIYYATGTCCTGGGTCCGACAGGCCCCTGGCAAGTCT RDSVKGRFTISRDNATTGGAATGGGTCGCCACCATCTCTGACGCTGGC KNSLYLQMNSLRDEDGGCTACATCTACTACCGGGACTCTGTGAAGGGC TAVYICARELPWRYAAGATTCACCATCAGCCGGGACAACGCCAAGAAC LDYWGQGTTVTVSSATCCCTGTACCTGCAGATGAACAGCCTGCGCGAC STKGPSVFPLAPSSKGAGGATACCGCCGTGTACATCTGTGCTAGAGAG STSGGTAALGCLVKDCTGCCTTGGAGATACGCCCTGGATTATTGGGGC YFPEPVTVSWNSGALCAGGGCACCACAGTGACCGTGTCCTCTGCTTCT TSGVHTFPAVLQSSGACCAAGGGACCCAGCGTGTTCCCTCTGGCTCCT LYSLSSVVTVPSSSLTCCAGCAAGTCTACCTCTGGCGGAACAGCTGCT GTQTYICNVNHKPSNCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAG TKVDKKVEPKSCDKTCCTGTGACAGTGTCCTGGAACTCTGGCGCTCTG HTCPPCPAPELLGGPACATCTGGCGTGCACACCTTTCCAGCAGTGCTG SVFLFPPKPKDTLMICAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTC SRTPEVTCVVVDVSHGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAG EDPEVKFNWYVDGVEACCTACATCTGCAACGTGAACCACAAGCCCTCC VHNAKTKPREEQYASAACACCAAGGTGGACAAGAAGGTGGAACCCAAG TYRVVSVLTVLHQDWTCCTGCGACAAGACCCACACCTGTCCTCCATGT LNGKEYKCKVSNKALCCTGCTCCAGAACTGCTGGGCGGACCCTCCGTG PAPIEKTISKAKGQPTTCCTGTTCCCTCCAAAGCCTAAGGACACCCTG REPQVYTLPPSREEMATGATCTCCCGGACCCCTGAAGTGACCTGCGTG TKNQVSLTCLVKGFYGTGGTGGATGTGTCCCACGAGGATCCCGAAGTG PSDIAVEWESNGQPEAAGTTCAATTGGTACGTGGACGGCGTGGAAGTG NNYKTTPPVLDSDGSCACAACGCCAAGACCAAGCCTAGAGAGGAACAG FFLYSKLTVDKSRWQTACgccTCCACCTACCGGGTGGTGTCCGTGCTG QGNVFSCSVMHEALHACCGTTCTGCACCAGGATTGGCTGAACGGCAAA NHYTQKSLSLSPGKGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTG (SEQ ID NO: 539)CCTGCCCCTATCGAAAAGACCATCTCTAAGGCC Linker GSGSGSGSGSGSGSGAAGGGCCAGCCCCGGGAACCTCAAGTGTACACC SGS TTGCCTCCCAGCCGGGAAGAGATGACCAAGAAC(SEQ ID NO: 529) CAGGTGTCCCTGACCTGCCTGGTTAAGGGCTTC scFv of VLDIVMTQSPLSLPVTP TACCCCTCCGATATCGCCGTGGAATGGGAGTCT 147 GEPASISCRSSKSLLAATGGCCAGCCTGAGAACAACTACAAGACCACA HSNGITYLYWYLQKPCCTCCTGTGCTGGACTCCGACGGCTCATTCTTC GQSPQLLIYQVSNLACTGTACTCCAAGCTGACCGTGGACAAGTCCAGA SGVPDRFSGSGSGTDTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTG FTLKISRVEAEDVGVATGCACGAGGCCCTGCACAATCACTACACCCAG YYCAQNLELFWTFGCAAGTCCCTGTCTCTGTCCCCTGGCAAAGGCTCC GTKVEIKRGGATCTGGTTCTGGATCCGGAAGCGGTTCTGGC (SEQ ID NO: 530)AGCGGCTCTGGATCTGACATCGTGATGACCCAG Linker GGGGSGGGGSGGGGSTCTCCACTGAGCCTGCCTGTGACACCTGGCGAG GGGS CCTGCTTCCATCTCCTGCCGGTCCTCTAAGTCC(SEQ ID NO: 531) CTGCTGCACTCTAACGGCATCACCTACCTGTAC VH EVQLVQSGAEVKKPGTGGTATCTGCAGAAGCCCGGCCAGTCTCCTCAG ASVKVSCKASGYTFTCTGCTGATCTACCAGGTGTCCAACCTGGCTTCT NYWLGWIKQAPGQCLGGCGTGCCCGATAGATTCTCCGGTAGCGGATCT EWIGDIYPGGDYINYGGAACCGACTTCACCCTGAAGATCTCCAGAGTG NEKFKGKATLTADTSGAAGCCGAGGACGTGGGCGTGTACTACTGTGCC ISTAYMELSRLRSDDCAGAACCTGGAACTGCCCTGGACCTTTGGCTGT TAVYYCARPNLPGDYGGCACCAAGGTGGAAATCAAGAGAGGCGGCGGA WGQGTTVTVSS*GGATCTGGCGGAGGTGGAAGCGGAGGCGGAGGA (SEQ ID NO: 532)AGCGGTGGCGGCGGATCTGAAGTTCAGTTGGTT CAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCTTCCGGCTAC ACCTTTACCAACTACTGGCTCGGCTGGATCAAGCAGGCCCCTGGACAGTGTCTGGAATGGATCGGC GACATCTACCCTGGCGGCGACTACATCAACTACAACGAGAAGTTCAAGGGCAAAGCTACCCTGACC GCCGACACCTCTATCTCCACCGCCTACATGGAACTGTCCCGGCTGAGATCTGACGACACCGCCGTG TACTATTGCGCCAGACCTAACCTGCCTGGCGACTATTGGGGCCAGGGCACAACAGTGACCGTGTCC TCTTAA (SEQ ID NO: 540) LightLight chain of B6 DIQMTQSPSSLSASV GACATCCAGATGACCCAGAGCCCTAGCAGCCTGchain GDRVTITCRASQDVT AGCGCTAGCGTGGGCGACAGGGTGACCATCACC PAVAWYQQKPGKAPKTGCAAGGCCAGCCAGGATGTGACCCCTGCCGTG LLIYSTSSRYTGVPSGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCC RFSGSGSGTDFTFTIAAGCTGCTGATCTACAGCACCAGCAGCAGGTAC SSLQPEDIATYYCQQACCGGCGTGCCCAGCAGGTTTAGCGGAAGCGGC HYTTPLTFGQGTKLEAGCGGCACCGACTTCACCTTCACCATCAGCAGC IKRTVAAPSVFIFPPCTGCAGCCCGAGGACATCGCCACCTACTACTGC SDEQLKSGTASVVCLCAGCAGCACTACACCACCCCTCTGACCTTCGGC LNNFYPREAKVQWKVCAGGGCACCAAGCTGGAGATCAAGAGAACCGTG DNALQSGNSQESVTEGCCGCTCCCTCCGTGTTCATCTTCCCACCATCT QDSKDSTYSLSSTLTGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTC LSKADYEKHKVYACEGTGTGCCTGCTGAACAACTTCTACCCTCGGGAA VTHQGLSSPVTKSFNGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTG RGEC*CAGTCCGGCAACTCCCAAGAGTCTGTGACCGAG (SEQ ID NO: 534)CAGGACTCCAAGGACAGCAGCTACTCCCTGTCC TCTACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCAC CAGGGACTGTCTAGCCCCGTGACCAAGTCCTTCAACAGAGGCGAGTGCTGA (SEQ ID NO: 535) B6x147(H3807)(bispecific antibody comprising the anti-PD-L1 B6 clone in igG formand the anti-LAG3 147(H3807) done in sc-Fv form) Heavy Heavy chain ofEVQLVESGGGLVQPG GAAGTGCAGCTGGTTGAATCTGGCGGCGGATTG Chain B6GSLRLSCAASGFTFS GCCGCCTCCGGCTTCACCTTCTCCAGCTACGAT SYDMSWVRQAPGKSLATGTCCTGGGTCCGACAGGCCCCTGGCAAGTCT EWVATISDAGGYIYYTTGGAATGGGTCGCCACCATCTCTGACGCTGGC RDSVKGRFTISRDNAGGCTACATCTACTACCGGGACTCTGTGAAGGGC KNSLYLQMNSLRDEDAGATTCACCATCAGCCGGGACAACGCCAAGAAC TAVYICARELPWRYATCCCTGTACCTGCAGATGAACAGCCTGCGCGAC LDYWGQGTTVTVSSAGAGGATACCGCCGTGTACATCTGTGCTAGAGAG STKGPSVFPLAPSSKCTGCCTTGGAGATACGCCCTGGATTATTGGGGC STSGGTAALGCLVKDCAGGGCACCACAGTGACCGTGTCCTCTGCTTCT YFPEPVTVSWNSGALACCAAGGGACCCAGCGTGTTCCCTCTGGCTCCT TSGVHTFPAVLQSSGTCCAGCAAGTCTACCTCTGGCGGAACAGCTGCT LYSLSSVVTVPSSSLCTGGGCTGCCTGGTCAAGGACTACTTTCCTGAG GTQTYICNVNHKPSNCCTGTGACAGTGTCCTGGARCTCTGGCGCTCTG TKVDKKVEPKSCDKTACATCTGGCGTGCACACCTTTCCAGCAGTGCTG HTCPPCPAPELLGGPCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTC SVFLFPPKPKDTLMIGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAG SRTPEVTCVVVDVSHACCTACATCTGCAACGTGAACCACAAGCCCTCC EDPEVKFNWYVDGVEAACACCAAGGTGGACAAGAAGGTGGAACCCAAG VHNAKTKPREEQYASTCCTGCGACAAGACCCACACCTGTCCTCCATGT TYRVVSVLTVLHQDWCCTGCTCCAGAACTGCTGGGCGGACCCTCCGTG LNGKEYKCKVSNKALTTCCTGTTCCCTCCAAAGCCTAAGGACACCCTG PAPIEKTISKAKGQPATGATCTCCCGGACCCCTGAAGTGACCTGCGTG REPQVYTLPPSREEMGTGGTGGATGTGTCCCACGAGGATCCCGAAGTG TKNQVSLTCLVKGFYAAGTTCAATTGGTACGTGGACGGCGTGGAAGTG PSDIAVLWESNGQPECACAACGCCAAGACCAAGCCTAGAGAGGAACAG NNYKTTPPVLDSDGSTACgccTCCACCTACCGGGTGGTGTCCGTGCTG FFLYSKLTVDKSRWQACCGTTCTGCACCAGGATTGGCTGAACGGCAAA QGNVFSCSVMHEALHGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTG NHYTQKSLSLSPGKCCTGCCCCTATCGAAAAGACCATCTCTAAGGCC (SEQ ID NO: 539)AAGGGCCAGCCCCGGGAACCTCAAGTGTACACC Linker GSGSGSGSGSGSGSGTTGCCTCCCAGCCGGGAAGAGATGACCAAGAAC SGS CAGGTGTCCCTGACCTGCCTGGTTAAGGGCTTC(SEQ ID NO: 529) GTTCAGCCTGGCGGATCTCTGAGACTGTCTTGT scFv of VLDIVMTQSPLSLPVTP CCTCCTGTGCTGGACTCCGACGGCTCATTCTTC 147(H3807)GEPASISCRSSKSLL CTGTACTCCAAGCTGACCGTGGACAAGTCTCGG HSQGITYLYWYLQKPTGGCAGCAGGGCAACGTGTTCTCCTGCTCTGTG GQSPQLLIYQVSNLAATGCACGAGGCCCTGCACAACCACTACACCCAG SGVPDRFSGSGSGTDAAGTCCCTGTCCCTGTCTCCCGGCAAAGGCTCC FTLKISRVEAEDVGVGGATCTGGTTCTGGATCCGGAAGCGGTTCTGGC YYCAQYLELPWTFGCAGCGGCTCTGGATCTGACATTGTGATGACCCAG GTKVEIKRAGCCCCCTGAGCCTCCCCGTGACCCCTGGAGAA (SEQ ID NO: 536)CCCGCCAGCATAAGCTGCAGATCCTCCAAAAGC Linker GGGGSGGGGSGGGGSCTGCTGCACTCCCAGGGAATAACCTACCTGTAT GGGGSTGGTACCTGCAGAAACCCGGCCAATCCCCCCAA (SEQ ID NO: 531)CTCCTGATATACCAAGTGTCCAACCTGGCCTCC VH EVQLVQSGAEVKKPGGGCGTGCCCGACAGATTCTCCGGCTCCGGCAGC ABVKVSCKASGYTFTGGTACCGACTTCACCCTCAAAATCTCCAGAGTG NYWLGWIKQAPGQCLGAAGCAGAAGACGTCGGCGTGTACTACTGCGCC EWIGDIYPGGDYIVYCAGTACCTGGAACTGCCCTGGACCTTCGGCtgt NEKFKGKATLTADTSGGCACCAAGGTGGAAATCAAGAGAGGCGGCGGA ISTAYMELSRLRSDDGGAAGCGGAGGCGGCGGTTCTGGTGGTGGCGGT TAVYYCARPNLPKDHAGCGGAGGTGGTGGATCTGAGGTGCAGCTGGTG WGQGTTVTVSS*CAGAGCGGAGCAGAGGTGAAGAAGCCAGGGGCC (SEQ ID NO: 537)AGCGTGAAGGTGAGCTGTAAGGCTAGTGGGTAC ACATTTACAAACTATTGGCTGGGATGGATTAAGCAGGCCCCAGGCCAAtgcCTGGAGTGGATAGGA GACATATACCCCGGAGGAGACTATATCGTGTACAACGAGAAGTTCAAGGGCAAGGCCACACTCACC GCTGATACARGCATCAGCACCGCCTACATGGAGCTGAGCCGACTGAGAAGCGACGACACAGCAGTG TATTACTGCGCCAGACCCAACCTGCCCAAGGACCACTGGGGACAAGGCACCACCGTGACCGTGAGC AGCtga (SEQ ID NO: 541) LightLight chain of B6 DIQMTQSPSSLSASV GACATCCAGATGACCCAGAGCCCTAGCAGCCTGchain GDRVTITCKASQDVT AGCGCTAGCGTGGGCGACAGGGTGACCATCACC PAVAWYQQRPGKAPKTGCAAGGCCAGCCAGGATGTGACCCCTGCCGTG LLIYSTSSRYTGVPSGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCC RFSGSGSGTDFTFTIAAGCTGCTGATCTACAGCACCAGCAGCAGGTAC SSLQPEDIATYYCQQACCGGCGTGCCCAGCAGGTTTAGCGGAAGCGGC HYTTPLTFGQGTKLEAGCGGCACCGACTTCACCTTCACCATCAGCAGC IKRTVAAPSVFIFPPCTGCAGCCCGAGGACATCGCCACCTACTACTGC SDEQLKSGTASVVCLCAGCAGCACTACACCACCCCTCTGACCTTCGGC LNNEYPREAKVOWKVCAGGGCACCAAGCTGGAGATCAAGAGAACCGTG DNALQSGNSQESVTEGCCGCTCCCTCCGTGTTCATCTTCCCACCATCT QDSKDSTYSLSSTLTGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTC LSKADYEKHKVYACEGTGTGCCTGCTGAACAACTTCTACCCTCGGGAA VTHQGLSSPVTKSFNGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTG RGEC*CAGTCCGGCAACTCCCAAGAGTCTGTGACCGAG (SEQ ID NO: 534)CAGGACTCCAAGGACAGCACCTACTCCCTGTCC TCTACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCAC CAGGGACTGTCTAGCCCCGTGACCAAGTCCTTCAACAGAGGCGAGTGCTGA (SEQ ID NO: 535)

TABLE 33Bispecific antibody comprising the anti-LAG3 clone in IgG form andthe anti-PD-L1 clone in scFvform (LAG3XPD-L1) Amino acid sequence(N′→C′) Nucleotide Sequence (5′→3′) 147xH12(bispecific antibody comprising the anti-LAG3 147 clone in gG formand the anti-PD-L1 H12 clone in scFv form) Heavy Heavy chain ofEVQLVQSGAEVKKPG GAGGTGCAGCTGGTGCAGAGCGGAGCAGAGGTG Chain 147ABVKVSCKASGYTFT AAGAAGCCAGGGGCCAGCGTGAAGGTGAGCTGT NYWLGWIKQAPGQGLAAGGCTAGTGGGTACACATTTACAAACTATTGG EWIGDIYPGGDYINYCTGGGATGGATTAAGCAGGCCCCAGGCCAAGGA NEKFKGKATLTADTSCTGGAGTGGATAGGAGACATATACCCCGGAGGA ISTAYMELSRLRSDDGACTATATCAATTACAACGAGAAGTTCAAGGGC TAVYYCARPNLPGDYAAGGCCACACTCACCGCTGATACAAGCATCAGC WGQGTTVTVSSASTKACCGCCTACATGGAGCTGAGCCGACTGAGAAGC GPSVFPLAPCSRSTSGACGACACAGCAGTGTATTACTGCGCCAGACCC ESTAALGCLVKDYFPAACCTGCCCGGCGACTACTGGGGACAAGGCACC EPVTVSWNSGALTSGACCGTGACCGTGTCTTCCgctAgcAccAAgggc VHTFPAVLQSSGLYSccctccgtgttccctctggccccAtgctcccgg LSSVVTVPSSSLGTKtccAcctccgAgtccAccgccgctctgggctgt TYTCNVDHKPSNTKVctggtgAAggActActtccctgAgcccgtgAcc DKRVESKYGPPCPPCgtgAgctggAActctggcgccctgAcctccggc PAPEFLGGPSVFLFPgtgcAcAccttccctgccgtgctgcAgtcctcc PKPKDTLMISRTPEVggcctgtActccctgtcctccgtggtgAccgtg TCVVVDVSQEDPEVQccttcctcctccctgggcAccAAgAcctAcAcc FNWYVDGVEVHNAKTtgcAAcgtggAccAcAAgccttccAAcAccAAg KPREEQFNSTYRVVSgtggAcAAgcgggtggAgtccAAgtAcggccct VLTVLHQDWLNGKEYccttgccctccctgccctgcccctgAgttcctg KCKVSNKGLPSSIEKggcggAccctccgtgttcctgttccctcctAAg TISKAKGQPREPQVYcctAAggAcAccctgAtgAtctcccggAcccct TLPPSQEEMTKNQVSgAggtgAcctgcgtggtggtggAcgtgtcccAg LTCLVKGFYPSDIAVgAAgAtcctgAggtccAgttcAAttggtAcgtg EWESNGQPENNYKTTgAtggcgtggAggtgcAcAAcgccAAgAccAAg PPVLDSDGSFFLYSRcctcgggAggAAcAgttcAActccAcctAccgg LTVDKSRWQEGNVFSgtggtgtctgtgctgAccgtgctgcAccAggAc CSVMHEALHNHYTQKtggctgAAcggcAAggAAtAcAAgtgcAAggtc SLSLSLGKAgcAAcAAgggcctgccctcctccAtcgAgAAA (SEQ ID NO: 542)AccAtctccAAggccAAgggccAgcctcgcgAg Linker GGGGSGGGGSGGGGScctcAggtgtAcAccctgcctcctAgccAggAA (SEQ ID NO: 543)gAgAtgAccAagAAtcAggtgtccctgAcAtgc scFv of VL DIQMTQSPSSLSASVctggtgAAgggcttctAcccttccgAtAtcgcc H12 GDRVTITCKASQDVTgtggAgtgggAgAgcAAcggccAgcCAgAgAAc PAVAWYQQKPGKAPKAActAcAAgAccAcccctcctgtgctggActcc LLIYTSSRITGVPSgAcggctccttcttcctgtActccAggctgAcc RFSGSGSGTDFTFTIgtggAcAAgtcccggtggcAggAAggcAAcgtc SSLQPEDIATYYCQQttttcctgctccgtgAtgcAcgAggccctgcAc HYTTPLTFGCGTKLEAAccActAcAcccAgAAgtccctgtccctgtct IKR ctgggcAAgGGTGGAGGTGGGTCTGGGGGTGGC(SEQ ID NO: 544) GGGTCAGGTGGAGGAGGTTCAGACATCCAGATG LinkerGGGGSGGGGSGGGGS ACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTG GGGGSGGCGACAGGGTGACCATCACCTGCAAGGCCAGC (SEQ ID NO: 531)CAGGATGTGACCCCTGCCGTGGCCTGGTACCAG VH EVQLVESGGGLVQPGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC GSLRLSCAASGFTFSTACAGCACCAGCAGCAGGTACACCGGCGTGCCC SYDMSWVRQAPGKCLAGCAGGTTTAGCGGAAGCGGCAGCGGCACCGAC EWVATISDAGGYIYYTTCACCTTCACCATCAGCAuCCTGCAGCCCGAG SDSVKGRETTSRDNAGACATCGCCACCTACTACTGCCAGCAGCACTAC KNSLYLQMNSLRDEDACCACCCCTCTGACCTTCGGCtgtGGCACCAAG TAVYICAREFGKRYACTGGAGATCAAGAGAGGTGGAGGCGGCTCAGGG LDYWGQGTTVTVSSGGGGGTGGATCAGGGGGAGGAGGATCAGGGGGA (SEQ ID NO: 545)GGCGGTAGTGAGGTGCAGCTGGTGGAGAGCGGA GGAGGACTGGTGCAACCCGGAGGCAGCCTGAGACTGAGCTGCGCTGCCAGCGGCTTCACCTTCAGC AGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAtgtCTGGAGTGGGTGGCCACCATCTCC GATGCGGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAAC GCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACATCTGC GCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGC AGCtga (SEQ ID NO: 546) LightLight chain of DIVMTQSPLSLPVTP GACATTGTGATGACCCAGAGCCCCCTGAGCCTC chain147 GEPASISCRSSKSLL CCCGTGACCCCTGGAGAACCCGCCAGCATAAGC HSNGITYLYWYLQKPTGCAGATCCTCCAAAAGCCTGCTGCACTCCAAC GQSPQLLIYQVSNLAGGAATAACCTAGCTGTATTGGTACCTGCAGAAA SGVPDRFSGSGSGTDCCCGGCCAATCCCCCCAACTCCTGATATACCAA FTLKISRVEAEDVGVGTGTCCAACCTGGCCTCCGGCGTGCCCGACAGA YYCAQNLELPWTFGGTTCTCCGGCTCCGGCAGCGGTACCGACTTCACC GTKVEIKRTVAAPSVCTCAAAATCTCCAGAGTGGAAGCAGAAGACGTC FIFPPSDEQLKSGTAGGCGTGTACTACTGCGCCCAGAATCTGGAACTG SVVCLLNNFYPREAKCCCTGGACCTTCGGCGGCGGCACCAAGGTGGAA VQWKVDNALQSGNSQATCAAGAGAACCGTGGCCGCTCCCTCCGTGTTC ESVTEQDSKDSTYSLATCTTCCCACCATCTGACGAGCAGCTGAAGTCC SSTLTLSKADYEKHKGGCACCGCTTCTGTCGTGTGCCTGCTGAACAAC VYACEVTHQGLSSPVTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAG TKSFNRGECGTGGACAATGCCCTGCAGTCCGGCAACTCCCAA (SEQ ID NO: 547)GAGTCTGTGACCGAGCAGGACTCCAAGGACAGC ACCTACTCCCTGTCCTCTACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCC TGCGAAGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGTCCTTCAACAGAGGCGAGTGCTGA (SEQ ID NO: 548) 147xB6(bispecific antibody comprising the anti-LAG3 147 clone in igG formand the anti-PD-L1 B6 clone in scFv form) Heavy Heavy chain ofEVQLVQSGAEVKKPG GAGGTGCAGCTGGTGCAGAGCGGAGCAGAGGTG Chain 147ASVKVSCKASGYTFT AAGAAGCCAGGGGCCAGCGTGAAGGTGAGCTGT NYWLGWIKQAPGQGLAAGGCTAGTGGGTACACATTTACAAACTATTGG EWIGDIYPGGDYINYCTGGGATGGATTAAGCAGGCCCCAGGCCAAGGA NEKFKGKATLTADTSCTGGAGTGGATAGGAGACATATACCCCGGAGGA ISTAYMELSRLRSDDGACTATATCAATTACAACGAGAAGTTCAAGGGC TAVYYCARPNLPGDYAAGGCCACACTCACCGCTGATACAAGCATCAGC WGQGTTVTVSSASTKACCGCCTACATGGAGCTGAGCCGACTGAGAAGC GPSVFPLAPCSRSTSGACGACACAGCAGTGTATTACTGCGCCAGACCC ESTAALGCLVKDYFPAACCTGCCCGGCGACTACTGGGGACAAGGCACC EPVTVSWNSGALTSGACCGTGACCGTGTCTTCCgctAgcAccAAgggc VHTFPAVLQSSGLYSccctccgtgttccctctggccccAtgctcccgg LSSVVTVPSSSLGTKtccAcctccgAgtccAccgccgctctgggctgt TYTCNVDHKPSNTKVctggtgAAggActActtccctgAgcccgtgAcc DKRVESKYGPPCPPCgtgAgctggAActctggcgccctgAcctccggc PAPEFLGGPSVFLFPgtgcAcAccttccctgccgtgctgcAgtcctcc PKPKDTLMISRTPEVggcctgtActccctgtcctccgtggtgAccgtg TCVVVDVSQEDPEVQccttcctcctccctgggcAccAAgAcctAcAcc FNWYVDGVEVHNAKTtgcAAcgtggAccAcAAgccttccAAcAccAAg KPREEQFNSTYRVVSgtggAcAAgcgggtggAgtccAAgtAcggccct VLTVLHQDWLNGKEYccttgccctccctgccctgcccctgAgttcctg KCKVSNKGLPSSIEKggcggAccctccgtgttcctgttccctcctAAg TISKAKGQPREPQVYcctAAggAcAccctgAtgAtctcccggAcccct TLPPSQEEMTKNQVSgAggtgAcctgcgtggtggtggAcgtgtcccAg LTCLVKGFYPSDIAVgAAgAtcctgAggtccAgttcAAttggtAcgtg EWESNGQPENNYKTTgAtggcgtggAggtgcAcAAcgccAAgAcCAAg PPVLDSDGSFFLYSRcctcgggAggAAcAgttcAActccAcctAccgg LTVDKSRWQEGNVFSgAgAtgAccAagAAtcAggtgtccctgAcAtgc CSVMHEALHNHYTQKctggtgAAgggcttctAcccttccgAtATCGCC SLSLSLGKAccAtctccAAggccAAgggccAgcctcgcgAg (SEQ ID NO: 542)cctcAggtgtAcAccctgcctcctAgccAggAA Linker GGGGSGGGGSGGGGSgAgAtgAccAagAAtcAGGtgtccctgAcAtgc (SEQ ID NO: 543)ctggtgAAgggcttctAcccttccgAtATCGCC scFv of VL DIQMTQSPSSLSASVGTGGAATGGGAGAGCAATGGCCAGCCTGAGAAC B6 GDRVTITCKASQDVTAACTACAAGACAACCCCTCCTGTGCTGGACTCC PAVAWYQQKPGKAPKGACGGCTCCTTCTTTCTGTACTCTCGCCTGACC LLIYSTSSRYTGVPSGTGGACAAGTCCAGATGGCAAGAGGGCAACGTG RFSGSGSGTDFTFTITTCTCCTGCTCCGTGATGCACGAGGCCCTGCAC SSLQPEDIATYYCQQAATCACTACACCCAGAAGTCCCTGTCTCTGTCC HYTTPLTFGCGTKLECTCGGAAAAGGCGGCGGAGGATCTGGCGGAGGC IKR GGTAGCGGTGGTGGCGGATCTGATATTCAGATG(SEQ ID NO: 544) ACCCAGTCTCCTTCCAGCCTGTCCGCTTCTGTG LinkerGGGGSGGGGSGGGGS GGCGACAGAGTGACCATCACATGCAAGGCCAGC GGGGSCAGGATGTGACCCCTGCTGTGGCTTGGTATCAG (SEQ ID NO: 531)CAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATC VH EVQLVESGGGLVQPGTACTCCACCTCCTCCAGATACACAGGCGTGCCC GSLRLSCAASGFTFSTCCAGATTCTCCGGCTCTGGCTCTGGCACCGAC SYDMSWVRQAPGKCLTTTACCTTTACAATCTCCAGCCTGCAGCCTGAG EWVATISDAGGYIYYGACATTGCCACCTACTACTGCCAGCAGCACTAC RDSVKGRFTISRDNAACCACACCTCTGACCTTTGGCTGCGGCACCAAG KNSLYLQMNSLRDEDCTGGAAATCAAGAGAGGTGGCGGAGGAAGCGGA TAVYICARELPWRYAGGCGGCGGTTCAGGTGGCGGTGGTTCAGGCGGT LDYWGQGTTVTVSS*GGTGGATCTGAAGTTCAGCTGGTGGAATCTGGC (SEQ ID NO: 549)GGCGGATTGGTTCAACCAGGCGGCTCTCTGAGA CTGTCTTGTGCCGCTTCCGGCTTCACCTTCTCCAGCTACGACATGTCCTGGGTCCGACAGGCCCCT GGAAAGTGTCTGGAATGGGTCGCCACCATCTCTGACGCTGGCGGCTACATCTACTACCGGGACTCT GTGAAGGGCAGATTCACCATCAGCCGGGACAATGCCAAGAACTCCCTGTACCTGCAGATGAACAGT CTGCGCGACGAGGACACCGCCGTGTACATCTGTGCTAGAGAGCTGCCTTGGCGCTACGCCCTGGAT TATTGGGGCCAGGGCACAACAGTGACAGTGTCCTCTTGA (SEQ ID NO: 550) Light Light chain of DIVMTQSPLSLPVTPGACATTGTGATGACCCAGAGCCCCCTGAGCCTC chain 147 GEPASISCRSSKSLLCCCGTGACCCCTGGAGAACCCGCCAGCATAAGC HSNGITYLYWYLQKPTGCAGATCCTCCAAAAGCCTGCTGCACTCCAAC GQSPQLLIYQVSNLAGGAATAACCTACCTGTATTGGTACCTGGAGAAA SGVPDRESGSGSGTLCCCGGCCAATCCCCCCAACTCCTGATATACCAA FTLKISRVEAEDVGVGTGTCCAACCTGGCCTCCGGCGTGCCCGACAGA YYCAQNLELPWTFGGTTCTCCGGCTCCGGCAGCGGTACCGACTTCACC GTKVEIKRTVAAPSVCTCAAAATCTCCAGAGTGGAAGCAGAAGACGTC FIFPPSDEQLKSGTAGGCGTGTAGTACTGCGCCCAGAATCTGGAACTG SVVCLLNNFYPREARCCCTGGACCTTCGGCGGCGGCACCAAGGTGGAA VQWKVDNALQSGNSQATCAAGAGAACCGTGGCCGCTCCCTCCGTGTTC ESVTEQDSKDSTYSLATCTTCCCACCATCTGACGAGCAGCTGAAGTCC SSTLTLSKADYEKHKGGCACCGCTTCTGTCGTGTGCCTGCTGAACAAC VYACEVTHQGLSSPVTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAG TKSFNRGECGTGGACAATGCCCTGCAGTCCGGCAACTCCCAA (SEQ ID NO: 547)GAGTCTGTGACCGAGCAGGACTCCAAGGACAGC ACCTACTCCCTGTCCTCTACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCC TGCGAAGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGTCCTTCAACAGAGGCGAGTGCTGA (SEQ ID NO: 548) 147(H3807)xH12(bispecific antibody comprising the anti-LAG3 147(H3807) clone in IgG formand the anti-PD-L1 HI 2 done in ScFvform) Heavy Heavy chain ofEVQLVQSGAEVKKPG GAGGTGCAGCTGGTGCAGAGCGGAGCAGAGGTG Chain 147(H3807)ASVKVSCKASGYTFT AAGAAGCCAGGGGCCAGCGTGAAGGTGAGCTGT NYWLGWIKQAPGQGLAAGGCCACACTCACCGCTGATACAAGCATCAGC EWIGDIYPGGDYIVYCTGGGATGGATTAAGCAGGCCCCAGGCCAAGGA NEKFKGKATLTADTSCTGGAGTGGATAGGAGACATATACCCCGGAGGA ISTAYMELSRLRSDDGACTATATCgtgTACAACGAGAAGTTCAAGGGC TAVYYCARPNLPKDHAAGGCCACACTCACCGCTGATACAAGCATCAGC WGQGTTVTVSSASTKACCGCCTACATGGAGCTGAGCCGACTGAGAAGC GPSVFPLAPCSRSTSGACGACACAGCAGTGTATTACTGCGCCAGACCC ESTAALGCLVKDYFPAACCTGCCCAAGGACCACTGGGGACAAGGCACC EPVTVSWNSGALTSGACCGTGACCGTGTCTTCCgctAgcAccAAgggc VHTFPAVLQSSGLYSccctccgtgttccctctggccccAtgctcccgg LSSVVTVPSSSLGTKtccAcctccgAgtccAccgccgctctgggctgt TYTCNVDHKPSNTKVctggtgAAggActActtccctgAgcccgtgAcc DKRVESKYGPPCPPCgtgAgctggAActctggcgccctgAcctccggc PAPEFLGGPSVFLFPgtgcAcAccttccctgccgtgctgcAgtcctcc PKPKDTLMISRTPEVggcctgtActccctgtcctccgtggtgAccgtg TCVVVDVSQEDPEVQccttcctcctccctgggcAccAAgAcctAcAcc FNWYVDGVEVHNAKTtgcAAcgtggAccAcAAgccttccAAcAccAAg KPREEQFNSTYRVVSgtggAcAAgcgggtggAgtccAAgtAcggccct VLTVLHQDWLNGKEYccttgccctccctgccctgcccctgAgttcctg KCKVSNKGLPSSIEKggcggAccctccgtgttcctgttccctcctAAg TISKAKGQPREPQVYcctAAggAcAccctgAtgAtctcccggAcccct TLPPSQEEMTKNQVSgAggtgAcctgcgtggtggtggAcgtgtcccAg LTCLVKGFYPSDIAVgAAgAtcctgAggtccAgttcAAttggtAcgtg EWESNGQPENNYKTTgAtggcgtggAggtgcAcAAcgccAAgAccAAg PPVLDSDGSFFLYSRcctcgggAggAAcAgttcAActccAcctAccgg LTVDKSRWQEGNVFSgtggtgtctgtgctgAccgtgctgcAccAggAc CSVMHEALHNHYTQKtggctgAAcggcAAggAAtAcAAgtgcAAggtc SLSLSLGKAgcAAcAAgggcctgccctcctccAtcgAgAAA (SEQ ID NO: 551)AccAtctccAAggccAAgggccAgcctcgcgAg Linker GGGGSGGGGSGGGGScctcAggtgtAcAccctgcctcctAgccAggAA (SEQ ID NO: 543)gAgAtgAccAagAAtcAggtgtccctgAcAtgc scFv of VL DIQMTQSPSSLSASVctggtgAAgggcttctAcccttccgAtAtcgcc H12 GDRVTITCKASQDVTgtggAgtgggAgAgcAAcggccAgccAgAgAAc PAVAWYQQKPGKAPKAActAcAAgAccAcccctcctgtgctggActcc LLIYSTSSRYTGVPSgAcggctccttcttcctgActccAggctgAcc RFSGSGSGTDFTFTIgtggAcAAgtcccggtggcAggAAggcAAcgtc SSLQPEDIATYYCQQttttcctgctccgtgAtgcAcgAggccctgcAc HYTTPLTFGCGTKLEAAccActAcAcccAgAAgtccctgtccctgtct IKR ctgggcAAgGGTGGAGGTGGGTCTGGGGGTGGC(SEQ LD NO: 544) GGGTCAGGTGGAGGAGGTTCAGACATCCAGATG LinkerGGGGSGGGGSGGGGS ACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTG GGGGSGGCGACAGGGTGACCATCACCTGCAAGGCCAGC (SEQ ID NO: 531)CAGGATGTGACCCCTGCCGTGGCCTGGTACCAG VH EVQLVESGGGLVQPGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC GSLRLSCAASGFTFSTACAGCACCAGCAGCAGGTACACCGGCGTGCCC SYDMSWVRQAPGKCLAGCAGGTTTAGCGGAAGCGGCAGCGGCACCGAC EWVATISDAGGYIYYTTCACCTTCACCATCAGCAGCCTGCAGCCCGAG SDSVKGRETISRDNAGACATCGCCACCTACTACTGCCAGCAGCACTAC KNSLYLQMNSLRDEDACCACCCCTCTGACCTTCGGCtgtGGCACCAAG TAVYICAREFGKRYACTGGAGATCAAGAGAGGTGGAGGCGGCTCAGGG LDYWGQGTTVTVSSGGGGGTGGATCAGGGGGAGGAGGATCAGGGGGA (SEQ ID NO: 545)GGCGGTAGTGAGGTGCAGCTGGTGGAGAGCGGA GGAGGACTGGTGCAACCCGGAGGCAGCCTGAGACTGAGCTGCGCTGCCAGCGGCTTCACCTTCAGC AGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAtgtCTGGAGTGGGTGGCCACCATCTCC GATGCGGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAAC GCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACATCTGC GCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGC AGCtga (SEQ ID NO: 552) LightLight chain of DIVMTQSPLSLPVTP GACATTGTGATGACCCAGAGCCCCCTGAGCCTC chain147(H3807) GEPASISCRSSKSLL CCCGTGACCCCTGGAGAACCCGCCAGCATAAGCHSQGITYLYWYLQKP TGCAGATCCTCCAAAAGCCTGCTGCACTCCcag GQSPQLLIYQVSNLAGGAATAACCTACCTGTATTGGTACCTGCAGPAA SGVPDRFSGSGSGTDCCCGGCCAATCCCCCCAACTCCTGATATACCAA FTLKISRVEAEDVGVGTGTCCAACCTGGCCTCCGGCGTGCCCGACAGA YYCAQYLELPWTFGGTTCTCCGGCTCCGGCAGCGGTACCGACTTCACC GTKVEIKRTVAAPSVCTCAAAATCTCCAGAGTGGAAGCAGAAGACGTC FIFPPSDEQLKSGTAGGCGTGTACTACTGCGCCCAGtacCTGGAACTG SVVCLLNNFYPREAKCCCTGGACCTTCGGCGGCGGCACCAAGGTGGAA VQWKVDNALQSGNSQATCAAGAGAACCGTGGCCGCTCCCTCCGTGTTC ESVTEQDSKDSTYSLATCTTCCCACCATCTGACGAGCAGCTGAAGTCC SSTLTLSKADYEKHKGGCACCGCTTCTGTCGTGTGCCTGCTGAACAAC VYACEVTHQGLSSPVTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAG TKSFNRGEC*GTGGACAATGCCCTGCAGTCCGGCAACTCCCAA (SEQ ID NO: 553)GAGTCTGTGACCGAGCAGGACTCCAAGGACAGC ACCTACTCCCTGTCCTCTACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCC TGCGAAGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGTCCTTCAACAGAGGCGAGTGCTGA (SEQ ID NO: 554) 147(H3807)xB6(bispecific antibody comprising the anti-LAG3 147(H2 3807) done in IgG formand the anti-PD-L1 B6 done in ScFv form) Heavy Heavy chain ofEVQLVQSGAEVKKPG GAGGTGCAGCTGGTGCAGAGCGGAGCAGAGGTG Chain 147(H3807)ASVKVSCKASGYTFT AAGAAGCCAGGGGCCAGCGTGAAGGTGAGCTGT NYWLGWIKQAPGQGLAAGGCTAGTGGGTACACATTTACAAACTATTGG EWIGDIYPGGDYIVYCTGGGATGGATTAAGCAGGCCCCAGGCCAAGGA NEKFKGKATLTADTSCTGGAGTGGATAGGAGACATATACCCCGGAGGA ISTAYMELSRLRSDDGACTATATCgtgTACAACGAGAAGTTCAAGGGC TAVYYCARPNLPKDHAAGGCCACACTCACCGCTGATACAAGCATCAGC WGQGTTVTVSSASTKACCGCCTACATGGAGCTGAGCCGACTGAGAAGC GPSVFPLAPCSRSTSGACGACACAGCAGTGTATTACTGCGCCAGACCC ESTAALGCLVKDYFPAACCTGCCCAAGGACCACTGGGGACAAGGCACC EPVTVSWNSGALTSGACCGTGACCGTGTCTTCCgctAgcAccAAgggc VHTFPAVLQSSGLYSccctccgtgttccctctggccccAtgctcccgg LSSVVTVPSSSLGTKtccAcctccgAgtccAccgccgctctgggctgt TYTCNVDHKPSNTKVctggtgAAggActActtccctgAgcccgtgAcc DKRVESKYGPPCPPCgtgAgctggAActctggcgccctgAcctccggc PAPEFLGGPSVFLFPgtgcAcAccttccctgccgtgctgcAgtcctcc PKPKDTLMISRTPEVggcctgtActccctgtcctccgtggtgAccgtg TCVVVDVSQEDPEVQccttcctcctccctgggcAccAAgAcctAcAcc FNWYVDGVEVHNAKTtgcAAcgtggAccAcAAgccttccAAcAccAAg KPREEQFNSTYRVVSgtggAcAAgcgggtggAgtccAAgtAcggccct VLTVLHQDWLNGKEYccttgccctccctgccctgcccctgAgttcctg KCKVSNKGLPSSIEKggcggAccctccgtgttcctgttccctcctAAg TISKAKGQPREPQVYcctAAggAcAccctgAtgAtctcccggAcccct TLPPSQEEMTKNQVSgAggtgAcctgcgtggtggtggAcgtgtcccAg LTCLVKGFYPSDIAVgAAgAtcctgAggtccAgttcAAttggtAcgtg EWESNGQPENNYKTTgAtggcgtggAggtgcAcAAcgccAAgAccAAg PPVLDSDGSFFLYSRcctcgggAggAAcAgttcAActccAcctAccgg LTVDKSRWQEGNVFSgtggtgtctgtgctgAccgtgctgcAccAggAc CSVMHEALHNHYTQKtggctgAAcggcAAggAAtAcAAgtgcAAggtc SLSLSLGKAgcAAcAAgggcctgccctcctccAtcgAgAAA (SEQ ID NO: 551)AccAtctccAAggccAAgggccAgcctcgcgAg Linker GGGGSGGGGSGGGGScctcAggtgtAcAccctgcctcctAgccAggAA (SEQ ID NO: 543)gAgAtgAccAagAAtcAggtgtccctgAcAtgc scFv of VL DIQMTQSPSSLSASVctggtgAAgggcttctAcccttccgAtATCGCC B6 GDRVTITCKASQDVTGTGGAATGGGAGAGCAATGGCCAGCCTGAGAAC PAVAWYQQKPGKAPKAACTACAAGACAACCCCTCCTGTGCTGGACTCC LLIYSTSSRYTGVPSGACGGCTCCTTCTTTCTGTACTCTCGCCTGACC RFSGSGSGTDFTFTIGTGGACAAGTCCAGATGGCAAGAGGGCAACGTG SSLQPEDIATYYCQQTTCTCCTGCTCCGTGATGCACGAGGCCCTGCAC HYTTPLTFGCGTKLEAATCACTACACCCAGAAGTCCCTGTCTCTGTCC IKR CTCGGAAAAGGCGGCGGAGGATCTGGCGGAGGC(SEQ ID NO: 544) GGTAGCGGTGGTGGCGGATCTGATATTCAGATG LinkerGGGGSGGGGSGGGGS ACCCAGTCTCCTTCCAGCCTGTCCGCTTCTGTG GGGGSGGCGACAGAGTGACCATCACATGCAAGGCCAGC (SEQ ID NO: 531)CAGGATGTGACCCCTGCTGTGGCTTGGTATCAG VH EVQLVESGGGLVQPGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATC GSLRLSCAASGFTFSTACTCCACCTCCTCCAGATACACAGGCGTGCCC SYDMSWVRQAPGKCLTCCAGATTCTCCGGCTCTGGCTCTGGCACCGAC EWVATISDAGGYIYYTTTACCTTTACAATCTCCAGCCTGCAGCCTGAG RDSVKGRFTISRDNAGACATTGCCACCTACTACTGCCAGCAGCACTAC KNSLYLQMNSLRDEDACCACACCTCTGACCTTTGGCTGCGGCACCAAG TAVYICARELPWRYACTGGAAATCAAGAGAGGTGGCGGAGGAAGCGGA LDYWGQGTTVTVSS*GGCGGCGGTTCAGGTGGCGGTGGTTCAGGCGGT (SEQ ID NO: 549)GGTGGATCTGAAGTTCAGCTGGTGGAATCTGGC GGCGGATTGGTTCAACCAGGCGGCTCTCTGAGACTGTCTTGTGCCGCTTCCGGCTTCACCTTCTCC AGCTACGACATGTCCTGGGTCCGACAGGCCCCTGGAAAGTGTCTGGAATGGGTCGCCACCATCTCT GACGCTGGCGGCTACATCTACTACCGGGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAAT GCCAAGAACTCCCTGTACCTGCAGATGAACAGTCTGCGCGACGAGGACACCGCCGTGTACATCTGT GCTAGAGAGCTGCCTTGGCGCTACGCCCTGGATTATTGGGGCCAGGGCACAACAGTGACAGTGTCC TCTTGA (SEQ ID NO: 555) LightLight chain of DIVMTQSPLSLPVTP GACATTGTGATGACCCAGAGCCCCCTGAGCCTC chain147(H3807) GEPASISCRSSKSLL CCCGTGACCCCTGGAGAKCCCGCCAGCATAAGCHSQGITYLYWYLQKP TGCAGATCCTCCAAAAGCCTGCTGCACTCCcag GQSPQLLIYQVSNLAGGAATAACCTACCTGTATTGGTACCTGCAGAAA SGVPDRFSGSGSGTDCCCGGCCAATCCCCCCAACTCCTGATATACCAA FTLKISRVEAEDVGVGTGTCCAACCTGGCCTCCGGCGTGCCCGACAGA YYCAQYLELPWTFGGTTCTCCGGCTCCGGCAGCGGTACCGACTTCACC GTKVEIKRTVAAPSVCTCAAAATCTCCAGAGTGGAAGCAGAAGACGTC FIFPPSDEOLKSGTAGGCGTGTACTACTGCGCCCAGtAcCTGGAACTG SVVCLLNNFYPREAKCCCTGGACCTTCGGCGGCGGCACCAAGGTGGAA VQWKVDNALQSGNSQATCAAGAGAACCGTGGCCGCTCCCTCCGTGTTC ESVTEQDSKDSTYSLATCTTCCCACCATCTGACGAGCAGCTGAAGTCC SSTLTLSKADYEKHKGGCACCGCTTCTGTCGTGTGCCTGCTGAACAAC VYACEVTHQGLSSPVTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAG TKSFNRGEC*GTGGACAATGCCCTGCAGTCCGGCAACTCCCAA (SEQ ID NO: 553)GAGTCTGTGACCGAGCAGGACTCCAAGGACAGC ACCTACTCCCTGTCCTCTACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCC TGCGAAGTGACCCACCAGGGACTGTCTAGCCCCGTGACGAAGTCCTTCAACAGAGGCGAGTGCTGA (SEQ ID NO: 554)

The constructed vectors were transiently expressed in ExpiCHO-S™ cells(Thermo Fisher, A29127) using (ExpiFectamine™CHO Kit, Thermo, A29129),cultured in ExpiCHO™ Expression medium (Thermo, A29100-01) under theconditions of 30 to 37° C. for 7 to 15 days in a CO₂ incubator equippedwith rotating shaker. Plasmid DNA (250 μg) and ExpiFectamin CHO Reagent(800 μL) were mixed with Opti-MEM® I medium (20 mL final volume) andallowed to stand at room temperature for 5 min. The mixed solution wasadded to 6×10⁶ ExpiCHO cells cultured in ExpiCHO Expression Medium andgently mixed in a shaker incubator at 37° C. with a humidifiedatmosphere of 8% CO₂ in air. At 18 hours post-transfection, 1.5 mL ofExpiFectamin CHO Transfection Enhancer 1 and 60 mL of ExpiFectamin CHOTransfection Feed were added to each flask.

Each BsAb was purified from the cell culture supernatant by recombinantProtein A affinity chromatography (Hitrap Mabselect Sure, GE Healthcare,28-4082-55) and gel filtration chromatography with a HiLoad 26/200Superdex200 prep grade column (GE Healthcare, 28-9893-36). SDS-PAGE(NuPage 4-12% Bis-Tris gel, NP0321) and size exclusion HPLC (Agilent,1200 series) analysis with SE-HPLC column (SWXL SE-HPLC column, TOSOH,G3000SWXL) were performed to detect and confirm the size and purity ofeach BsAb. Purified proteins were concentrated in PBS by ultrafiltrationusing a Amicon Ultra 15 30K device (Merck, UFC903096), and proteinconcentrations were estimated using a nanodrop (Thermo, Nanodrop One).When a two-vector system is applied, the ratio between light to heavychain could be 1:1 to 1:3 by weight. Alternatively, a one-vector systemthat contains both chains in one single vector can also be used.

The prepared anti-PD-L1/anti-LAG3 bispecific antibodies are named asH12x147, H12x147(H3807), B6x147, and B6x147(H3807), 147xH12,147(H3807)xH12, 147xB6, and 147(H3807)xB6, respectively, wherein theformer refers to the clone in the IgG form and the latter refers to theclone in the scFv form.

Example 4. Characterization of Bispecific Antibodies H12x147 and 147xH12

4.1. Binding of the Bispecific Antibodies

To evaluate the binding activity to PD-L1 and LAG3 of the bispecificantibodies (BsAb; H12x147 and 147xH12) prepared in Example 3, the BsAbwere subjected to ELISA test. Briefly, microtiter plates were coatedwith each of human PD-L1-Fc protein (Sinobio, 10084-H02H1 and humanLAG3-His protein (Sinobio, 16498-H08H) at 0.5 μg/ml in PBS, 100 μl/wellat 4° C. overnight, then blocked with 100 μl/well of 5% BSA. Four-folddilutions of each of the BsAbs starting from 100 nM were added to eachwell and incubated for 1-2 hours at RT. The plates were washed withPBS/Tween and then incubate with goat-anti-human IgG antibody conjugatedwith Horse Radish Peroxidase (HRP) (Pierce, cat #31413) for 1 hour atRT. After washing, the plates were developed with TMB substrate andanalyzed by spectrophotometer at OD 450-630 nm. The results are shown inFIG. 33. As shown in FIG. 33, all the BsAbs tested can bind to both ofhuman PD-L1 and human LAG3 proteins with high activities.

4.2. Binding Affinity of Bispecific Antibodies

The binding affinities of bispecific antibodies PD-L1 and LAG3 of thebispecific antibodies (BsAb; 147xH12, 147H3807xB6 and B6x147H3807)prepared in Example 3 to PD-L1 protein and human LAG3 protein weretested with BIACORE™ using a capture method.

The results are shown in Table 34.

TABLE 34 Antibody Human PD-L1 (KD (M)) Human LAG3 (KD (M)) 147xH122.74E−08 1.35E−08 147H3807xB6 5.94E−09 1.63E−09 B6x147H3807 1.18E−098.87E−09

As shown in Table 34 and FIG. 33, the bispecific antibody tested displayrelatively high binding affinities to both of human PD-L1 and human LAG3proteins.

In addition, SEE assay was conducted, and the obtained results are shownin FIG. 34, the results indicating that the bispecific antibody testedinhibits the binding between MHC II and LAG3, thereby increasing T cellactivity by MHC II and TCR.

4.3. Activity of the Bispecific Antibodies to Promote Human T CellImmune Response

To test the ability of bispecific antibodies to stimulated T cellresponse, Jurkat cell activation assay was used. Jurkat cellstransfected with human Lag3 and Pd1 by lentivirus were used as theresponder cells. Raji cells which overexpressed PDL1 was used as theantigen presenting cells (APC). Staphylococcal enterotoxins E (SEE) aresuperantigen, which was used as the stimulator in this assay. In thissystem, ectopically expressed huLAG3 and huPD-1 can suppress SEstimulated IL-2 production by Jurkat cells, while anti-LAG3 andanti-PD-L1 antibodies can reverse IL-2 production. In short, Raji(1×10⁴) were co-cultured with Jurkat T cells (1×10⁵) in the presence ofsuperantigen. Bispecific antibodies and their counterpart monoantibodies(starting from 100 nM diluted for 6 dose) were added to the mixedculture. 48 hrs later, supernatant was collected for IL2 production. Asshown in FIG. 34 (upper panel), bispecific antibodies (147xH12 (labeledas 147-H12) and H12X147 (labeled as H12-147)) can dose dependentlypromote I12 production by Jurkat cells.

To further evaluate in vitro function of bispecific antibodies towardsprimary T cells, mixed lymphocyte reaction was performed. Humandendritic cells (DCs) were differentiated from CD14+ monocytes in thepresence of GM-CSF and IL-4 for 7 days. CD4+ T cells isolated fromanother donor were then co-cultured with the DCs and serially dilutedantibodies. 5 days after mixed culture, the culture supernatant wasassayed for IFNγ production. The results in FIG. 34 (lower panel)indicated that both bispecific antibodies (147XH12 (labeled as A3L1) andH12X147 (labeled as L1A3) can significantly promote IFNγ production.

4.4. Tumor Growth Inhibition of the Bispecific Antibodies (In VivoAssay)

Double humanized mice that express the extracellular domain of humanPD-1 and human LAG3 were used. Mouse colon adenocarcinoma cells (MC38)were engineered to express human PD-L1. Double humanized mice(hLAG3/hPD-1) were subcutaneously implanted with 5×10⁵ MC38-hPD-L1 cellson day 0. On day 10, mice with an average tumor volume of 137 mm³ wereselected and randomized into four treatment groups (N=7/group). Mousewere intraperitoneally administered isotype control (5 mg/kg), H12(anti-PD-L1 antibody, 5 mg/kg), 147H (anti-LAG3 antibody, 5 mg/kg) and147xH12 (6.6 mg/kg) every other day for 8 doses, starting from day 10.Tumor volumes were monitored by caliper measurement twice per week forthe duration of the experiment (29 days). Neither H12 nor 147H showedtumor inhibition at 5 mg/kg. By contrast, 147xH12 demonstrated robustinhibition of MC38 tumor growth, with a TGI of 67.7% at the end of thestudy (FIG. 35).

Example 5. Characterization of Bispecific Antibodies 147xH12 and147(H3807)xH12

5.1. Binding of the Bispecific Antibodies

To evaluate the binding activity to LAG3 of the bispecific antibodies(BsAb; 147xH12 and 147(H3807)xH12) prepared in Example 3, the BsAbs weresubjected to ELISA test. Briefly, microtiter plates were coated withhuman LAG3-His protein (Sinobio, 16498-H08H1 at 0.5 μg/ml in PBS, 100μl/well at 4° C. overnight, then blocked with 100 μl/well of 5% BSA.Four-fold dilutions of each of the BsAbs starting from 100 nM were addedto each well and incubated for 1-2 hours at RT. The plates were washedwith PBS/Tween and then incubate with goat-anti-human IgG antibodyconjugated with Horse Radish Peroxidase (HRP) (Pierce, cat #31413) for 1hour at RT. After washing, the plates were developed with TMB substrateand analyzed by spectrophotometer at OD 450-630 nm. As shown in FIG. 33,the BsAbs 147(H3807)xH12 displays more improved binding activity tohuman LAG3 protein.

5.2. Activity of the Bispecific Antibodies to Promote Human T CellImmune Response

The effect of bispecific antibodies prepared in Example 3 was furtherstudied using PBMCs from healthy donors. In brief, human DCs weredifferentiated from CD14+ monocytes for 7 days. Purified CD4+ T cellsisolated from another donor was stimulated by anti-CD3/CD28 for 2 days.Serially diluted antibodies were then added to DC and T cell co-culturein the presence of superantigen and incubated for 5 days and the culturemedium was collected for IL-2 level. As showed in FIG. 36, bispecificantibodies could significantly stimulate IL-2 production in primary CD4+T cells, which was superior than combination of their correspondingmonoantibodies. Data are shown as mean values from triplicate wells±SD.

Moreover, the effect of bispecific antibodies prepared in Example 3 wasstudied using PBMCs from healthy donors. In brief, human DCs weredifferentiated from CD14+ monocytes for 5 days, followed by LPStreatment for maturation. Pan T cells were isolated from another donorPBMC. Serially diluted antibodies were then added to mature DC and Tcell co-culture and incubated for 5 days and the culture medium wascollected for IFNγ level. As showed in FIG. 37, bispecific antibodiescould significantly stimulate IFNγ production in primary pan T cells,which was superior than combination of their correspondingmonoantibodies. Data are shown as mean values from duplicate wells±SD.

5.3. Developability of Bispecific Antibodies

The developability regarding the physicochemical properties to PD-L1 andLAG-3 bispecific antibodies (BsAb; B6x147H3807 and 147(H3807)xB6) wasassessed. The quality attributes for the BsAbs were evaluated by severalanalytical methods. Briefly, the purity was measured by Sizeexclusion-high performance liquid chromatography (SE-HPLC) and both ofthe BsAbs showed the high purity over 99%. The thermal stability byProtein thermal shift (PTS) with fluorescence labeled Realtime-polymerase chain reaction (RT-PCR) was analyzed. Their meltingtemperature was observed over 67° C. which indicated that the testarticles have stable structural integrity. To evaluate solubility of themolecules, the proteins were concentrated to 20 mg/mL usingultrafiltration (Amicon Ultra-15 spin concentrator). As a result, thevisible particles were not observed by visual inspection and noincrement of aggregates was confirmed by SE-HPLC. The Isoelectric point(pI) of each bsabs measured by capillary isoelectric focusing (cIEF)were 8.26 and 8.35, respectively. This pI range is appropriate toproceed downstream process and formulation development. Overall, asshown in Table 19. It showed that the tested BsAbs (B6x147H3807 and147(H3807)xB6) have proper physicochemical properties for the successfuldevelopment.

TABLE 35 Content Method B6x147H3807 147(H3807)xB6 Purity SEC 99.8 99.8Thermal PTS 61.8 62.0 Stability 77.7 71.6 Solubility Visual Easy toconcentrate Easy to concentrate inspection up to 20 mg/mL, clear up to20 mg/mL, clear pI cIEF 8.56 7.65

Example 6. The Effect of B3807 on Inhibition of the Binding of FGL1 toLAG3

This example tested the anti-LAG3 antibody B3807's activity ininhibiting the binding between LAG3 and Fibrinogen-like Protein 1(FGL1).

It was recently reported that Fibrinogen-like Protein 1 (FGL1) isanother functional ligand of LAG3, apart from MHC-1l (Cell. 2019;176:1-14). FGL-1 is secreted from liver and highly produced by cancercells. FGL-1 inhibits antigen-specific T cell activation and inversely,blockade of FGL-1 potentiates anti-tumor response. Interaction betweenFGL-1 and LAG3 may represent another mechanism for immune evasion.

Recombinant FGL-1 were coated on a 96 well plated at a concentration of1 μg/ml and incubated overnight at 4° C. Serially diluted anti-LAG3antibody B3807 (starting from 10 μg/ml and 1:3 dilution) andbiotin-labeled LAG3-ECD (2 μg/ml) were incubated with FGL-1 coated wellsat room temperature for 2 hours. After extensive washing with the washbuffer, streptavidin-HRP was added. As shown in FIG. 47, B3807dose-dependently inhibited the binding of FGL-1 to LAG3 protein.

The present disclosure is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the disclosure, and any compositions or methodswhich are functionally equivalent are within the scope of thisdisclosure. It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present disclosure without departing from the spirit or scope ofthe disclosure. Thus, it is intended that the present disclosure coverthe modifications and variations of this disclosure provided they comewithin the scope of the appended claims and their equivalents.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference

1. An anti-PD-L1/anti-LAG3 bispecific antibody, comprising an anti-PD-L1antibody or an antigen-binding fragment thereof and an anti-LAG3antibody or an antigen-binding fragment thereof, wherein the anti-PD-L1antibody or antigen-binding fragment thereof is capable of specificallybinding to an immunoglobulin C (Ig C) domain of a human Programmeddeath-ligand 1 (PD-L1) protein, wherein the Ig C domain consists ofamino acid residues 133-225; and the anti-LAG3 antibody orantigen-binding fragment thereof comprises a VH CDR1 having an aminoacid sequence selected from the group consisting of SEQ ID NOS: 116-117,354, and 453-460; a VH CDR2 having an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 118-119, 355, and 461-467; a VH CDR3having an amino acid sequence selected from the group consisting of SEQID NOs: 120-160, 356, and 468-475; a VL CDR1 having an amino acidsequence selected from the group consisting of SEQ ID NOS: 163-195, 229,357, and 490; a VL CDR2 having an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 196-217, 358, and 476-483; and a VL CDR3having an amino acid sequence selected from the group consisting of SEQID NOS: 218-228, 230-253, 359, and 484-489.
 2. The anti-PD-L1/anti-LAG3bispecific antibody of claim 1, wherein the anti-PD-L1 antibody orantigen-binding fragment thereof is capable of binding to at least oneof amino acid residues Y134, K162, or N183 of the PD-L1 protein. 3.(canceled)
 4. The anti-PD-L1/anti-LAG3 bispecific antibody of claim 1,wherein the anti-PD-L1 antibody or antigen-binding fragment thereof doesnot bind to an immunoglobulin V (Ig V) domain of the PD-L1 protein,wherein the Ig V domain consists of amino acid residues 19-127. 5.(canceled)
 6. The anti-PD-L1/anti-LAG3 bispecific antibody of claim 1,comprising an anti-PD-L1 antibody or an antigen-binding fragment thereofand an anti-LAG3 antibody or an antigen-binding fragment thereof,wherein the anti-PD-L1 antibody or antigen-binding fragment thereofcomprises: (1) a VH CDR1 having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 and SEQ ID NO: 61-67; (2) a VH CDR2having an amino acid sequence selected from the group consisting of SEQID NO: 2, SEQ ID NO: 68-77, and 525-527; (3) a VH CDR3 having an aminoacid sequence selected from the group consisting of SEQ ID NO: 3, SEQ IDNO: 78-90 and SEQ ID NO: 513-519; (4) a VL CDR1 having an amino acidsequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO:91-92, and SEQ ID NO: 520-521; (5) a VL CDR2 having an amino acidsequence selected from the group consisting of SEQ ID NO: 5 and SEQ IDNO: 93-105; and (6) a VL CDR3 having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 6, SEQ ID NO: 106-111, and SEQID NO: 522-524, and the anti-LAG3 antibody or antigen-binding fragmentthereof comprises: (i) a VH CDR1 having an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 116-117, 354, and 453-460; (ii)a VH CDR2 having an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 118-119, 355, and 461-467; (iii) a VH CDR3having an amino acid sequence selected from the group consisting of SEQID NOs: 120-160, 356, and 468-475; (iv) a VL CDR1 having an amino acidsequence selected from the group consisting of SEQ ID NOS: 163-195, 229,357, and 490; (v) a VL CDR2 having an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 196-217, 358, and 476-483; and (vi)a VL CDR3 having an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 218-228, 230-253, 359, and 484-489. 7-8.(canceled)
 9. The anti-PD-L1/anti-LAG3 bispecific antibody of any-eneclaim 6, wherein the anti-PD-L1 antibody or antigen-binding fragmentthereof comprises a heavy chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NOS: 7-26, 113,493, 495, 497, 499, 501, 503, 505, 507, 509, and 511, or a polypeptidehaving at least 90% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 7-26, 113, 493, 495, 497, 499,501, 503, 505, 507, 509, and
 511. 10. The anti-PD-L1/anti-LAG3bispecific antibody of any-ene claim 6, wherein the anti-PD-L1 antibodyor antigen-binding fragment thereof comprises a light chain variableregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 27-33, 494, 496, 498, 500, 502, 504, 506, 508,510, and 512, or a peptide having at least 90% sequence identity to anamino acid sequence selected from the group consisting of SEQ ID NOS:27-33, 494, 496, 498, 500, 502, 504, 506, 508, 510, and
 512. 11. Theanti-PD-L1/anti-LAG3 bispecific antibody of any-ene claim 6, wherein theanti-LAG3 antibody or antigen-binding fragment thereof comprises a heavychain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 254-302, 352, 360-373, 375, 377,379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405,407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433,435, 437, 439, 441, 443, 445, 447, 449, 451 and 491, or a polypeptidehaving at least 90% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 254-302, 352, 360-373, 375,377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403,405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431,433, 435, 437, 439, 441, 443, 445, 447, 449, 451 and
 491. 12. Theanti-PD-L1/anti-LAG3 bispecific antibody of any-ene claim 6, wherein theanti-LAG3 antibody or antigen-binding fragment thereof comprises a lightchain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 303-351, 353, 374, 376, 378, 380,382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408,410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436,438, 440, 442, 444, 446, 448, 450, 452 and 492, or a peptide having atleast 90% sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 303-351, 353, 374, 376, 378, 380, 382,384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410,412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438,440, 442, 444, 446, 448, 450, 452 and
 378. 13. (canceled)
 14. A methodfor treating a disease associated with PD-L1, LAG3, or both thereof,comprising administering to the patient a composition comprising theanti-PD-L1/anti-LAG3 bispecific antibody of claim 1 and apharmaceutically acceptable carrier.
 15. The method of claim 14, whereinthe disease associated with PD-L1, LAG3, or both thereof is cancer orinfection.
 16. The method of claim 15, wherein the cancer is a solidtumor.
 17. The method of claim 15, wherein the cancer is selected fromthe group consisting of bladder cancer, liver cancer, colon cancer,rectal cancer, endometrial cancer, leukemia, lymphoma, pancreaticcancer, small cell lung cancer, non-small cell lung cancer, breastcancer, urethral cancer, head and neck cancer, gastrointestinal cancer,stomach cancer, oesophageal cancer, ovarian cancer, renal cancer,melanoma, prostate cancer and thyroid cancer. 18-28. (canceled)